Comparison of murine retroorbital plexus and facial vein blood collection to mitigate animal ethics issues / 한국실험동물학회지

Background@#Blood collection is an important procedure used in animal experiments. Blood collection methods that reduce pain, injury, and stress in experimental animals are important with regard to animal ethics. Various comparative studies of blood collection methods have been reported; however, there are no comparative studies on serial blood collection considering animal ethics. To suggest simple methods that minimize pain during serial blood collection, we compared the retroorbital plexus (RP) and facial vein (FV) blood collection methods performed by both experienced and novice groups. The experienced and novice groups collected up to 0.4 mL of blood via the RP and FV methods every second day for 2 weeks. After blood collection, all mice were evaluated by corticosterone concentrations for stress, hematological, immunological, and histological analyses. @*Results@#We found that the FV methods reduced the collection time, pain, distress, tissue damage and lasting harms without anesthesia. Corticosterone concentrations in the peripheral blood were decreased in mice subjected FV methods compare with those subjected to RP methods. The proportion of granulocytes and monocytes, such as macrophages in the peripheral blood and spleen, was decreased in mice subjected to FV methods compared with that in mice subjected to RP methods in both experienced and novice groups. White blood cells were infiltrated in RP areas with severe tissue damage and inflammation. @*Conclusions@#With respect to animal ethics, we suggest that the FV method, a simple and fast technique that can easily be performed by both experienced and novice researchers, is suitable for serial blood collection.

Late Recurrence in a Rectal Cancer Patient Who Underwent Preoperative Chemoradiotherapy Followed by Local Excision: A Case Report

Some patients who have undergone preoperative chemoradiotherapy (CRT) following surgery have been diagnosed with late recurrence more than 5 years after treatment, raising questions about the possible benefit extending surveillance beyond the recommended 5 years. In 2011, a 71-year-old male patient was diagnosed with T3N+ low-lying rectal cancer located 3 cm from the anal verge before undergoing long-course preoperative CRT. After CRT, the patient was reexamined and diagnosed with ycT1–2N0 lesion, so local excision (LE) was performed. The patient underwent intensive surveillance for up to 5 years, and no evidence of recurrence was found. At 74 months after surgery, the patient was hospitalized for a hematochezia, and local recurrence at the excision site and peritoneal seeding nodules were identified. Considering the late recurrence in this patient, it might be necessary to long-term follow-up beyond 5 years in patients with preoperative CRT followed by LE.

Late Recurrence in a Rectal Cancer Patient Who Underwent Preoperative Chemoradiotherapy Followed by Local Excision: A Case Report

Some patients who have undergone preoperative chemoradiotherapy (CRT) following surgery have been diagnosed with late recurrence more than 5 years after treatment, raising questions about the possible benefit extending surveillance beyond the recommended 5 years. In 2011, a 71-year-old male patient was diagnosed with T3N+ low-lying rectal cancer located 3 cm from the anal verge before undergoing long-course preoperative CRT. After CRT, the patient was reexamined and diagnosed with ycT1–2N0 lesion, so local excision (LE) was performed. The patient underwent intensive surveillance for up to 5 years, and no evidence of recurrence was found. At 74 months after surgery, the patient was hospitalized for a hematochezia, and local recurrence at the excision site and peritoneal seeding nodules were identified. Considering the late recurrence in this patient, it might be necessary to long-term follow-up beyond 5 years in patients with preoperative CRT followed by LE.

Comparison of murine retroorbital plexus and facial vein blood collection to mitigate animal ethics issues / 한국실험동물학회지

Background@#Blood collection is an important procedure used in animal experiments. Blood collection methods that reduce pain, injury, and stress in experimental animals are important with regard to animal ethics. Various comparative studies of blood collection methods have been reported; however, there are no comparative studies on serial blood collection considering animal ethics. To suggest simple methods that minimize pain during serial blood collection, we compared the retroorbital plexus (RP) and facial vein (FV) blood collection methods performed by both experienced and novice groups. The experienced and novice groups collected up to 0.4 mL of blood via the RP and FV methods every second day for 2 weeks. After blood collection, all mice were evaluated by corticosterone concentrations for stress, hematological, immunological, and histological analyses. @*Results@#We found that the FV methods reduced the collection time, pain, distress, tissue damage and lasting harms without anesthesia. Corticosterone concentrations in the peripheral blood were decreased in mice subjected FV methods compare with those subjected to RP methods. The proportion of granulocytes and monocytes, such as macrophages in the peripheral blood and spleen, was decreased in mice subjected to FV methods compared with that in mice subjected to RP methods in both experienced and novice groups. White blood cells were infiltrated in RP areas with severe tissue damage and inflammation. @*Conclusions@#With respect to animal ethics, we suggest that the FV method, a simple and fast technique that can easily be performed by both experienced and novice researchers, is suitable for serial blood collection.

Association of Body Composition with Long-Term Survival inNon-metastatic Rectal Cancer Patients / Journal of the Korean Cancer Association, 대한암학회지

Purpose@#We evaluated the association of body composition with long-term oncologic outcomes innon-metastatic rectal cancer patients. @*Materials and Methods@#We included 1,384 patients with stage(y)0-III rectal cancer treated at Asan Medical Centerbetween January 2005 and December 2012. Body composition at diagnosis was measuredusing abdomino-pelvic computed tomography (CT). Sarcopenia, visceral obesity (VO), andsarcopenic obesity (SO) were defined using CT measured parameters such as skeletal muscleindex (total abdominal muscle area, TAMA), visceral fat area (VFA), and VFA/TAMA. Inflammatorystatus was defined as a neutrophil-lymphocyte ratio of ! 3. Obesity was categorizedby body mass index (! 25 kg/m2). @*Results@#Among the 1,384 patients, 944 (68.2%) had sarcopenia and 307 (22.2%) had SO. The5-year overall survival (OS) rate was significantly lower in sarcopenic patients (no sarcopeniavs. sarcopenia; 84% vs. 78%, p=0.003) but the 5-year recurrence-free survival (RFS) ratewas not different (77.3% vs. 77.9% p=0.957). Patients with SO showed lower 5-year OS(79.1% vs. 75.5% p=0.02) but no difference in 5-year RFS (p=0.957). Sarcopenia, SO, VO,and obesity were not associated with RFS. However, obesity, SO, age, sex, inflammatorystatus, and tumor stage were confirmed as independent factors associated with OS on multivariateanalysis. In subgroup analysis, association of SO with OS was more prominent inpatients with (y)p stage 0-2 and no inflammatory status. @*Conclusion@#The presence of SO and a low body mass index at diagnosis are negatively associated withOS in non-metastatic rectal cancer patients.

Early Postoperative Anastomotic Obstruction Due to an Intraluminal Blood Clot After Laparoscopic Anterior Resection: A Case Report

Early postoperative anastomotic obstruction after colorectal surgery rarely develops. Herein, we present a case of a 50-year-old healthy woman who had an early postoperative anastomotic obstruction which was revealed caused by a blood clot and successfully managed by endoscopic approach. The patient was discharged after laparoscopic anterior resection and visited the emergency department one day after because of abdominal pain. Computed tomography showed that the anastomosis site was obstructed with low-density material. Intraoperative endoscopy was performed under general anesthesia and blood clot filling the lumen were identified. As the scope was advanced to the blood clot with air inflation, the blood clot was evacuated. The anastomosis site could be obstructed by blot clot with mucous debris albeit it is a rare condition. An endoscopic approach seems to be the first option in the diagnosis and treatment of postoperative obstruction at the anastomosis site and it could prevent unnecessary laparotomy.

Evaluation of infertility efficacy of the E. coli expressed STF2-GnRH vaccine in male cats

Gonadotropin-releasing hormone (GnRH) is secreted from the hypothalamus and anti-GnRH antibodies are not formed under normal conditions. However, administration an excess of recombinant GnRH protein results in the formation of anti-GnRH. We evaluated the efficacy of the recombinant Salmonella typhimurium flagellin fljB (STF2)-GnRH vaccine in inducing infertility in 17 intact male cats. The first vaccination and a boosting vaccine was injected for examination. Serum was obtained from blood collected at monthly intervals and anti-GnRH antibodies and testosterone concentrations were determined. Six months after the vaccination, testicular samples are obtained and used for histological examination. Compared with sham control group, the injection groups showed an increase in anti-GnRH antibody titers and testosterone concentrations tended to be reduced in the injection groups and increased in the control group. Histological evaluations and Johnsen's testicular biopsy scores revealed testicular hypoplasia in the 2 injection groups. Consequently, normal sexual maturation with sperm production was observed in the control group. In contrast, the cats that received the GnRH vaccine showed weak (2 of 7 cats) or moderate (4 out of 7 cats) dose-dependent infertility effects. On the basis of the results, the STF2-GnRH vaccine was identified to be effective in inducing infertility in male cats. The results of this study thus indicate the possibility of immunological castration targeting feral cats.

Data analysis for improving population management in animal shelters in Seoul / 대한수의학회지

A total of 11,395 animals were impounded in shelters in Seoul in 2013. The Animal Protection Division of the Seoul metropolitan government has annual contracts with local veterinary associations as well as Korean animal rescue and management organizations for providing shelter to animals, and collects monthly statistics from these groups. In 2013, the collected intake and outcome data for 25 districts were reviewed to analyze shelter capacity in terms of housing capacity (monthly daily average intake, required holding capacity, and adoption-driven capacity), staff capacity (staff hours required for daily care), and live release rate. Seasonal variations in the monthly daily average intake were observed, indicating that management of these shelters requires various strategies. This study was performed to analyze and interpret meaningful statistics for improving the efficiency of animal shelters in Seoul. However, inconsistent collection of animal statistics limited data compilation. Creation of a basic animal statistics matrix with reference to well-designed matrices from recognized professional animal shelters is essential. These complied statistical data will help plan for future animal shelter needs in Seoul.

Quantitative measurement of influenza virus replication using consecutive bronchoalveolar lavage in the lower respiratory tract of a ferret model

The ferret is an established animal model of influenza virus infection. Although viral replication in the upper respiratory tract is usually measured with consecutively collected nasal washes, daily evaluation of viral replication in the lung is limited because a large numbers of ferrets need to be sacrificed at consecutive time points. To overcome this limitation, we performed a virus quantification assay using bronchoalveolar lavage (BAL) fluid. This non-invasive BAL technique allows consecutive quantification of virus replication in the lungs of living ferrets. Our method can be used for the longitudinal evaluation of virus tropism in the lower respiratory tract.

A survey of the use of veterinary anesthetics in Korea / 대한수의학회지

This study was conducted to investigate actual conditions associated with veterinary anesthetic drug use in Korea, and to obtain responses from Korean veterinarians and researchers pertaining to the use of anesthetic drugs. To accomplish this, a nationwide survey was issued to veterinarians working at animal hospitals and to researchers in the Korean Association for Laboratory Animal Science (KALAS). A self-administered questionnaire-based survey was then conducted in which respondents were asked questions about actual conditions associated with the use of animal anesthetic drugs. The survey revealed that the distribution and management of animal medicines in Korea was quite vulnerable to misuse or abuse due to a variety of factors. Therefore, a relevant regulatory system should be strictly enforced to protect vulnerable individuals from abuse or misuse.

Kinetics of proinflammatory cytokines after intraperitoneal injection of tribromoethanol and a tribromoethanol/xylazine combination in ICR mice / 한국실험동물학회지

Tribromoethanol (2,2,2-tribromoethanol, TBE) is a popular injectable anesthetic agent used in mice in Korea. Our goal was to assess the risks associated with side effects (lesions) in the abdominal cavity, especially at high doses. To understand the underlying pathophysiological changes, we examined levels of cytokines through ELISA of abdominal lavage fluid and spleen collected from mice treated with low and high-dose TBE. ICR mice were anesthetized using one of the following protocols: a combination of TBE 200 mg/kg (1.25%) and xylazine 10 mg/kg; TBE 400 mg/kg (1.25%); and TBE 400 mg/kg (2.5%). Administration of high-dose TBE (400 mg/kg) increased the interleukin-1beta and interleukin-6 levels in the peritoneal cavity over the short term (<1 day) compared with sham controls and low-dose TBE (200 mg/kg) groups. Cytokine expression in the low-dose TBE group was similar to the control group, whereas in the high-dose TBE group cytokine levels were higher in abdominal lavage fluid and spleen over the long term (10 days post-injection). We conclude that a combination of TBE 200 mg/kg (1.25%) and xylazine (10 mg/kg) is a safe and effective anesthetic for use in animals.

Identification of bacteria from the oral cavity and cloaca of snakes imported from Vietnam / 한국실험동물학회지

Reptiles are used for various purposes these days, including public exhibits, medicinal applications, and as laboratory animals. As the international exchange of reptiles has gradually increased, more people have had the opportunity to come in contact with these animals. Snakes typically live in the rhizosphere where various bacterial strains exist and as such they can lead to opportunistic human diseases. When snakes are encountered in veterinary medicine, it is necessary to monitor their microflora. Native microflora of reptiles imported from other countries has not yet been reported in Korea. In this study, oral and cloacae samples were collected from 18 Burmese pythons transported from Vietnam. The specimens were incubated at 37degrees C for 18 h to produce colony growth under aerobic condition and isolated colonies were then identified using a VITEK automated identification system. There were fourteen types of aerobic bacteria isolated from both oral and cloacae samples, nine from only oral specimens, and fifteen from only cloacae specimens. Most bacteria isolated were opportunistic pathogens of humans which therefore have the potential to induce disease in people. Based on the microflora and the prevalence of bacterial strains in snakes, quarantine procedures for reptiles transported internationally should be strengthened. Characterization of the microflora of reptiles with the potential to induce zoonosis should be performed in those used as laboratory animals and to prevent zoonotic outbreaks in the general population as well as among veterinarians.

Isolation and Expression Profile of the Ca(2+)-Activated Chloride Channel-like Membrane Protein 6 Gene in Xenopus laevis / 한국실험동물학회지

To clone the first anion channel from Xenopus laevis (X. laevis), we isolated a calcium-activated chloride channel (CLCA)-like membrane protein 6 gene (CMP6) in X. laevis. As a first step in gene isolation, an expressed sequence tags database was screened to find the partial cDNA fragment. A putative partial cDNA sequence was obtained by comparison with rat CLCAs identified in our laboratory. First stranded cDNA was synthesized by reverse transcription polymerase-chain reaction (RT-PCR) using a specific primer designed for the target cDNA. Repeating the 5' and 3' rapid amplification of cDNA ends, full-length cDNA was constructed from the cDNA pool. The full-length CMP6 cDNA completed via 5'- and 3'-RACE was 2,940 bp long and had an open reading frame (ORF) of 940 amino acids. The predicted 940 polypeptides have four major transmembrane domains and showed about 50% identity with that of rat brain CLCAs in our previously published data. Semi-quantification analysis revealed that CMP6 was most abundantly expressed in small intestine, colon and liver. However, all tissues except small intestine, colon and liver had undetectable levels. This result became more credible after we did real-time PCR quantification for the target gene. In view of all CLCA studies focused on human or murine channels, this finding suggests a hypothetical protein as an ion channel, an X. laevis CLCA.

Evaluation on Efficacy and Safety of Tribromoethanol and Tribromoethanol plus alpha2-Adrenergic Agonists in Different Mouse Strains / 한국실험동물학회지

The present study was carried out to provide a guideline for injecting tribromoethanol (TBE) as the main anesthetic agent, while adjusting the doses of xylazine (X) and medetomidine (M) according to different strains of mice (male ICR, C57BL/6, and BALB/c). Seven intraperitoneal injection anesthesia protocols using TBE and mixtures of TBE and alpha2-adrenergic agonists (TBE/X and TBE/M) were compared in terms of their efficacy and safety (anesthetic duration, death rate, and the development of pathological lesions of abdominal organs). All animals that were injected with a low dose of TBE (200 mg/kg) displayed clear signs of light anesthesia with a strong pedal withdrawal reflex. Despite the good anesthetic effect, a high dose of TBE (400 mg/kg) was not a suitable anesthetic for major surgery in all mouse strains because of the risk of pathologic changes in the abdominal organs, such as retention of the digestive tract, peritonitis, and fibrinoid adhesion. TBE200/X10 and TBE200/M0.5 (TBE, 200 mg/kg; X, 10 mg/kg; M, 0.5 mg/kg) appeared to be safe and provided satisfactory anesthesia in ICR mice. Finally, there were clear differences in anesthetic efficacy among ICR, C57BL/6, and BALB/c strains. TBE/M and TBE/X did not anesthetize BALB/c mice, and it anesthetized C57BL/6 mice for a short time. When administered with TBE/X and TBE/M maintained the sedation of ICR mice. We were able to establish different regimes for each strain (TBE200/X20 for C57BL/6, TBE300/X10 and TBE200/M1 for BALB/c). Our results showed that TBE/X and TBE/M could be recommended as an anesthetic mixture, with the dose appropriately adjusted according to mouse strain.

Evaluation on Efficacy and Safety of Tribromoethanol and Tribromoethanol plus alpha2-Adrenergic Agonists in Different Mouse Strains / 한국실험동물학회지

The present study was carried out to provide a guideline for injecting tribromoethanol (TBE) as the main anesthetic agent, while adjusting the doses of xylazine (X) and medetomidine (M) according to different strains of mice (male ICR, C57BL/6, and BALB/c). Seven intraperitoneal injection anesthesia protocols using TBE and mixtures of TBE and alpha2-adrenergic agonists (TBE/X and TBE/M) were compared in terms of their efficacy and safety (anesthetic duration, death rate, and the development of pathological lesions of abdominal organs). All animals that were injected with a low dose of TBE (200 mg/kg) displayed clear signs of light anesthesia with a strong pedal withdrawal reflex. Despite the good anesthetic effect, a high dose of TBE (400 mg/kg) was not a suitable anesthetic for major surgery in all mouse strains because of the risk of pathologic changes in the abdominal organs, such as retention of the digestive tract, peritonitis, and fibrinoid adhesion. TBE200/X10 and TBE200/M0.5 (TBE, 200 mg/kg; X, 10 mg/kg; M, 0.5 mg/kg) appeared to be safe and provided satisfactory anesthesia in ICR mice. Finally, there were clear differences in anesthetic efficacy among ICR, C57BL/6, and BALB/c strains. TBE/M and TBE/X did not anesthetize BALB/c mice, and it anesthetized C57BL/6 mice for a short time. When administered with TBE/X and TBE/M maintained the sedation of ICR mice. We were able to establish different regimes for each strain (TBE200/X20 for C57BL/6, TBE300/X10 and TBE200/M1 for BALB/c). Our results showed that TBE/X and TBE/M could be recommended as an anesthetic mixture, with the dose appropriately adjusted according to mouse strain.

Antifungal Effects of New Synthetic Materials, KAF-200522 and KAF-200522-HCl, on in vitro and in vivo Models / 한국실험동물학회지

KAF-200522 and its chloride form, KAF-200522-HCl, were invented in Chemon inc. as new triazole antifungal agents with excellent activities in vivo and in vitro against wide range of fungi. As a result of in vitro susceptibility measurements, 80% minimum inhibitory concentrations (MIC80) of both test articles against Candida albican sp. and Aspergillus fumigatus sp. were below 0.0156 microg/mL, which were over 4,100 times lower than those of fluconazole against fluconazole resistant C. albican sp. and A. fumigatus sp., and were over 16 times lower than those of amphotericin B against above same fungi. Additionally, against representative dermatophytes, Trichophyton sp., the MIC80s of both test articles were below 0.0156 microg/mL which were over 64 times lower than those of fluconazole and amphotericin B. As in vivo antifungal activities in A. fumigatus sp. infected mouse models, KAF-200522 treatment group at 600 mg/kg showed 80% survival rate which was 2 times higher than that of amphotericin B and showed 13.7 days in the mean survival time (MST) which was about 2.1 times higher than that of amphotericin B. But in KAF-200522-HCl treatment groups, all animals were found dead in contrast to 40% survival rate in amphotericin B treatment group, however dose dependent increases in MST was revealed. In conclusion, antifungal activities of KAF-200522 and its mimics, KAF-200522-HCl in vitro and in vivo were confirmed in this study, therefore the potentiality of the present compounds to be developed into new antifungal drug was expected.

Laboratory Rodents Negatively Affected by Construction Environment / 한국실험동물학회지

This report describes rodents in a laboratory animal facility that was adversely affected by a noisy environment during construction work. There was much noise and vibration as well as dust caused by the drilling and hammering. The noise levels, frequencies, and length of time when occurring in the drilling and hammering, were all measured. The drilling showed noise levels ranging from 50-90 decibels (dB) (A-filter, A), and the hammering presented 60-70 dB (A). Some researchers raised problems regarding animal experiments, including skin injuries resulted from self-mutilation, and increase of mortality. This gives useful information to people who plan to renovate laboratory animal facilities as it is a very rare case.

The Effect of Intravenous Lipo-Prostaglandin E1 Injectioin in a Rat Foraminal Stenosis Model / 대한통증학회지

BACKGROUND: Lipo-prostaglandin E1 (Lipo-PGE1) has vasodilating and platelet aggregation inhibitory characteristics and it has been used as a treatment for patients with blood flow dysfunction disease. Based on the mechanisms of lumbar spinal stenosis, including veno congestion, neuro-ischemia and mechanical compression, we aimed to study whether intravenous Lipo-PGE1 injection has any therapeutic effect on hyperalgesia in a rat foraminal stenosis model. METHODS: In this study, twenty male Sprague-Dawley rats were divided into the control (n = 10) and Lipo-PGE(1)(n = 10) groups. A small stainless steel rod was inserted into the L5-6 intervertebral foramen to induce intervertebral foramen stenosis and chronic DRG compression. In the Lipo-PGE1 group, 0.15micron g/kg of Lipo-PGE(1) were injected intravenously via a tail vein for 10 days starting from the 3rd day after operation. Behavioral testing for mechanical and thermal hyperalgesia was performed for 3 weeks after the injections. RESULTS: From the 10th day after Lipo-PGE(1) injection, the rats in the experimental group showed significant recovery of their mechanical threshold, and this effect was maintained for 3 weeks. No significant differences of the thermal hyperalgesia were observed between the two groups. CONCLUSIONS: These findings suggest that intravenously injected Lipo-PGE1 may be effective for alleviating neuropathic pain, which isthe main symptom of spinal stenosis, by improving the blood flow dysfunction.

Protective Effects of Trimetazidine in a Rabbit Model of Transient Spinal Cord Ischemia / 대한흉부외과학회지

BACKGROUND: Paraplegia remains unresolved as the most dreaded operative complication with surgical treatment of descending thoracic and thoracoabdominal aortic diseases. In this study, the neuroprotective effect of trimetazidine that has been used clinically for ischemic heart disease was investigated in a rabbit spinal cord ischemia model. MATERIAL AND METHOD: Thirty-three New Zealand white rabbits were randomized as follows: control group undergoing abdominal aortic occlusion but receiving no pharmacologic intervention(Group 1, n=17); TMZ group(Group 2, n=16) receiving 3 mg/kg trimetazidine intravenously before the occlusion of the aorta. Ischemia was induced by clamping the abdominal aorta just distal to the left renal artery for 30 minutes. Neurologic status was assessed at 2, 24, and 48 hours after the operation according to the modified Tarlov scale, then the lumbosacral spinal cord was processed for histopathologic examinations 48 hours after the final assessment. RESULT: The average motor function score was significantly higher in the TMZ group(3.20 +/- 0.77 vs 1.13 +/- 1.25 at 2 hours, 3.50 +/- 0.76 vs 1.45 +/- 1.57 at 24 hours, and 3.91 +/- 0.30 vs 1.86 +/- 1.86 at 48 hours after operation; p value

Protective Effects of Trimetazidine in a Rabbit Model of Transient Spinal Cord Ischemia / 대한흉부외과학회지

BACKGROUND: Paraplegia remains unresolved as the most dreaded operative complication with surgical treatment of descending thoracic and thoracoabdominal aortic diseases. In this study, the neuroprotective effect of trimetazidine that has been used clinically for ischemic heart disease was investigated in a rabbit spinal cord ischemia model. MATERIAL AND METHOD: Thirty-three New Zealand white rabbits were randomized as follows: control group undergoing abdominal aortic occlusion but receiving no pharmacologic intervention(Group 1, n=17); TMZ group(Group 2, n=16) receiving 3 mg/kg trimetazidine intravenously before the occlusion of the aorta. Ischemia was induced by clamping the abdominal aorta just distal to the left renal artery for 30 minutes. Neurologic status was assessed at 2, 24, and 48 hours after the operation according to the modified Tarlov scale, then the lumbosacral spinal cord was processed for histopathologic examinations 48 hours after the final assessment. RESULT: The average motor function score was significantly higher in the TMZ group(3.20 +/- 0.77 vs 1.13 +/- 1.25 at 2 hours, 3.50 +/- 0.76 vs 1.45 +/- 1.57 at 24 hours, and 3.91 +/- 0.30 vs 1.86 +/- 1.86 at 48 hours after operation; p value