Epidemiologic and clinical studies have related acute and, less frequently, chronic life stress to cardiovascular diseases. In addition, animal models suggest that chronic psychological stress could cause atherosclerosis, probably by increasing sympathetic activation. In this cross-sectional study we evaluated the association between job strain, as one of the markers of workplace stress, and the urinary excretion of Vanilmandelic acid (VMA) upon awakening as a sympathoadrenal activity marker in the morning. Subjects were 936 male and 823 female civil servants working in departments related to the municipality of a city in Toyama Prefecture, Japan, in the spring of 2001. VMA was measured by high-performance liquid chromatography. We found that there was an age dependent increase in the level of VMA and females had higher VMA levels than males. Males who were current smokers had significantly lower VMA levels than nonsmokers after adjusting for age. Job strain level did not relate to VMA concentration in urine after adjusting for age and smoking status both in men and women. In addition, working and sleeping hours as predictor variables were also not associated with urinary VMA levels upon awakening in the morning. In conclusion, it seems that job strain does not independently relate to the sympathoadrenal activity, but the interaction between job strain and other variables such as personal characteristics and environmental factors and their relation with sympathoadrenal activity should further be explored. The effects of acute stress on heart disease are well supported by epidemiologic studies investigating natural life stressors.4-5 In contrast to chronic stress, however, acute stress is easier to model and can be studied under controlled laboratory conditions in both humans and animals.Anecdotal reports and case studies6-10 Although many aspects of one’s work environment have been studied, much interest has focused on models of inherent tension at work and its relation to the development of CAD. One such model is the ‘job strain’ model proposed by Karasek,11,12 where job strain is defined as jobs with high demand but low decision latitude (control). Although the studies regarding presence of stress at work and subsequent CAD development have been largely positive6-9,13 suggesting a causal relationship between this form of chronic stress and development of CAD, the pathophysiologic pathway through which work related stress causes CAD has not been fully addressed.Work related stress is the most widely studied chronic life stress in relation to coronary artery diseases (CAD).14 However, little is known about the long lasting effects of psychosocial stress at work on sympathoadrenal activity, which might be a pathologic burden for CAD incidence in humans.15-17 Thus, this study was conducted to evaluate the impact of the job strain model and its dimensions (job demand and control) on vanilmandelic acid (VMA) as a metabolite to mark sympathoadrenal activity among Japanese civil servants. An increase in excretion of VMA upon awakening in the morning was expected with increasing level of job strain. According to previous reports,18,19 VMA excretion is less changed by various quantities of foods, in addition, due to the number of our study population we decided to measure one sympathoadrenal marker rather than assessing adrenaline and noradrenaline together.An extensive body of evidence from animal models revealed that chronic psychological stress could lead, probably via a mechanism involving excessive sympathetic nervous system activation, to exacerbation of coronary artery atherosclerosis as well as to transient endothelial dysfunction and even necrosis.The target population for this cross-sectional study was all the 2180 civil servants working in the departments related to the municipality of a city in Toyama Prefecture, Japan, in the spring of 2001. The response rate for questionnaire survey was 80.7%  were men and 823(46.8%) were women). We also collected urine samples upon awakening in the morning for VMA measurement. The response rate for VMA measurement was 74.9% (1634 subjects). The overall response rate for those subjects with data on both questionnaire and urinary VMA level reached to 70.8%  were men and 721 (46.7%) were women).20 which was set up to investigate the degree and causes of the social gradient in morbidity and mortality in a cohort of civil servants in London.This follow up study is in collaboration with the Whitehall II study,21 1) administrative; 2) professional; 3) clerical; and 4) protective, transportation and telecommunication service workers which we put them under the category of office support. We also stratified the age of the participants into four groups: <29, 30-39, 40-49 and 50+ years of age. Usual working and sleeping hours per day during the month prior to the study plus the smoking habit was asked through a self-reported questionnaire.Civil servants were classified into four groups based on the job classification system used in the census of the national survey:7 to report the work environment characteristics (based on Karasek’s job strain model) in the Whitehall II study was adopted for this study to assess job strain (see appendix). Face and content validity plus internal consistency-reliability (see appendix) was satisfactory for the scale, which had already been used in other fields in Japan and also in other countries. We created the four job strain groups constructing a high and low group for demand and control dimensions by dividing the distributions of these dimensions at their respective medians and cross-classifying subjects. The questionnaire used by Bosma22 with Kawaguchi’s23 analyzer at BML company in Saitama, Japan. The urine samples of 40 subjects were divided and analyzed in separate days to assess reproducibility of the measurement. The function of the assay was found to be satisfactory as its intraassay coefficient of variation was 1.11 and the mean difference between the measurements was 0.03 . The VMA level was adjusted for the creatinine concentration in the urine and expressed as mg/g.creatinine (mg/g.cre).The first morning urine specimen (5-6ml) upon awakening was collected by the subject themselves at their home. Each collection tube contained 0.8ml of 6N hydrochloric acid to acidify the urine to a pH less than 1; the urine samples were collected upon arrival of the subjects at the annual health check-up place and kept in iceboxes before being refrigerated at -80°C until analysis. VMA was analyzed with HPLC by modified method of PisanoWe performed univariate analysis using a t-test and a one-way analysis of variance (ANOVA) to compare the mean values of VMA for socio-demographic characteristics and smoking status of the subjects. In addition, a three-way ANOVA was used to detect the differences in VMA among the four groups of the job strain model, adjusting for age and smoking status in both sexes. To test whether there is a multivariate association between working and sleeping hours plus job control and demand with VMA, we conducted a multiple linear regression analysis with VMA as the dependant variable. A two-tailed p value of less than 0.05 was set as the significance level. Data were analyzed using SPSS.Mean values of VMA did not show any significant difference among the four groups of the job strain model after adjusting for age and smoking habits of the civil servants in both men and women .β coefficient=0.087, p<0.05 for men; standardized β coefficient=0.338, p<0.001 for women). Other independent variables including sleeping hours, working hours, job demand and control did not associate with the urinary levels of VMA. Partial correlation coefficients between VMA and demand/control ratio were -0.01 and 0.04 for men and women respectively, after adjusting for age.Results of the multiple regression analysis  show thaMeanwhile, VMA levels for current smokers (mean±SE: 1.75±0.06) were significantly lower than nonsmokers’ (mean±SE: 1.93±0.05) after adjusting for age in male civil servants . The sam4 There is also evidence that overreactivity of the sympathetic nervous system plays a role in the development of hypertension, particularly in its early stages.24 Epidemiologic investigations of variations in heart rate have revealed a relation between increased sympathetic tone and long commuting time or extensive overtime.25 Park et al.26 found a slight positive association between working hours and low frequency power of heart rate variability and urinary excretion of adrenaline in a study of 238 male engineers in Korea. In another study conducted by Sasaki et al.27 on 147 subjects, they reported that the group with longer working hours (68 hours/ week) had lower urinary noradrenaline level for one age group (30-39 years) than the group with shorter working hours. In both studies, however, the measurements were conducted through the usual working day with the knowledge that different levels of acute stress in the hours prior to assessment could divert the results to either direction.Animal models revealed that chronic psychosocial stress could lead, probably via a mechanism involving excessive sympathetic nervous system activation, to exacerbate atherosclerosis.28-30 due to the ability for controlling research condition.Acute impact of psychosocial stress on increasing sympathoadrenal activity has been addressed more frequently in the literature31) as our questionnaire was based on the actual sleeping hours in the last month prior to the study. However, the large size of our study population could compensate the impact of those who changed their usual sleeping hours in the night prior to urine collection.In our study on a large number of civil servants we found no relationship between morning urinary level of VMA upon awakening and the level of job strain. In addition, working hours in a month prior to measurement did not show a significant association with VMA level. It should be mentioned that although excretion of VMA is less changed by the quantity of diet taken in a day prior to assessment, we could not control the results for the sleeping hours prior to urinary collection  of monoamine oxidase,39 an enzyme which metabolizes catecholamines, this evidence can also explain the lower VMA level in smokers.Forearm venous plasma noradrenaline concentration increases with age.40 reported that platelet monoamine oxidase manifested a significant increase in females compared to males, which may be the cause for the significant difference in the urinary VMA levels between men and women in all age groups of our study population.VeralIn conclusion, despite the limitations in our cross-sectional study and single measurement of only one sympathetic activity marker, we showed that there was not an association between job strain and urinary VMA levels upon awakening in the morning in Japanese civil servants.


Positive clinical outcomes of a group of surviving puppies from a litter affected by parvoviral myocarditis are detailed in this case report. Past reports focus on the negative outcomes of littermates of puppies who have died of parvoviral myocarditis. In this case, two puppies in a shelter setting, from a litter exposed to parvovirus, died suddenly with parvoviral myocarditis diagnosed at necropsy. The other seven puppies were screened for cardiac health with echocardiogram, electrocardiogram, and cardiac troponin I prior to adoption. All seven puppies had normal echocardiograms, electrocardiograms, and normal initial and recheck cardiac troponin I results. At recheck 2 years after the initial round of testing, two of the puppies were screened and continue to have normal cardiac diagnostics. All seven dogs are alive and thriving at 5 years old in homes with adopters who were given a complete medical history on the dogs prior to adoption. In summary, the outcomes for puppies in litters affected by parvoviral myocarditis are variable but they do not have to be grave. The use of cardiac diagnostics including echocardiogram, electrocardiogram and cardiac troponin I may serve as a prognostic basis for assessing the potential outcomes for the surviving puppies in affected litters. The present case report describes a litter of puppies who were admitted to an animal shelter who had a history of perinatal exposure to parvovirus. Two of the littermates died suddenly secondary to parvoviral myocarditis (PMC). The seven surviving littermates were evaluated for evidence of cardiac dysfunction through echocardiography, electrocardiography (ECG), and cardiac troponin I (cTnI) prior to adoption. The puppies evaluated have since survived almost 5 years.Canine parvovirus 2 (CPV-2) is a non-enveloped DNA virus that has been reported to cause both severe enteritis and myocarditis in dogs –5. PuppiPrevious literature suggests a grave prognosis for puppies within litters affected by PMC. Entire litters or large portions of litters affected by myocarditis were reported to have poor outcomes including sudden death in the weeks after birth or a decline into heart failure in the months after birth. Information about longer term positive outcomes for puppies in affected litters is very limited, leaving a gap in information concerning long-term outcomes , 11–15.The objectives of reporting this case are to demonstrate (1) the use of echocardiography, ECG, and cTnI as potential prognostic indicators for surviving littermates of puppies who have died of PMC, (2) that survival is variable and prognosis may be good for littermates of puppies with PMC, and (3) that despite being fully informed of the potential for poor prognosis, people were still willing to adopt these puppies.Eight large mixed breed puppies (A1–A8) from a litter of 10 were transported to a rescue group in early June 2018 after one pup from their litter died acutely (A9). The litter was initially part of a larger group of dogs admitted to an animal shelter including two intact males (Dog C and D) and two intact females (Dog A and B). Dog A was actively nursing the litter of ten puppies, six females (A1–A5) and four males (A6–A10), estimated to be one to two weeks old (A1–A10) .No clinical signs of illness were reported for any of the 10 puppies (A1–A10) who were examined by lay staff at shelter admission (Day 0). The four adults  were also examined on Day 0. Clinical signs reported were similar in all adults and included vomiting, diarrhea, and lethargy consistent with CPV-2 infection. Rectal swab samples from all dogs and puppies in the group were tested individually at intake with a lateral flow immunochromatographic (LFI) test for CPV-2 antigen. Dogs A, B, C and D tested positive for CPV-2 antigen, while puppies A1–A10 tested negative.Dog B was euthanized the day of intake due to the severity of clinical signs and submitted for necropsy which confirmed severe parvoviral enteritis. The remaining three adult dogs and puppies were kept separated from the general population. Adults were treated with supportive care, and recovered uneventfully . None ofOne puppy, A10, was taken from the shelter without record and was lost to follow up. Puppies were monitored daily and were reportedly thriving in the shelter. Routine preventative care was provided including the parental administration of a modified live Distemper-Adenovirus-Parvovirus-Parainfluenza vaccine on Day 27.No clinical signs of illness were reported in the litter until A9 was found deceased 34 days after intake. A repeat LFI parvovirus antigen test returned a negative result from the rectal swab of A9.Bilateral pulmonary congestion was noted on postmortem examination of A9 at University of Kentucky Veterinary Diagnostic Laboratory. Upon histopathologic examination, multifocal to coalescing regions of the myocardium were expanded by minimal to small streams of collagenous connective tissue that was infiltrated with low to moderate numbers of lymphocytes and plasma cells. Large, basophilic, intranuclear viral inclusion bodies were multifocally located in cardiomyocytes . FindingBecause previous literature suggested a potentially grave prognosis for the remaining littermates a transfer was arranged to facilitate foster care and assessment by the author. Cardiac work-up was planned if the puppies were deemed healthy on initial physical exam.Post-transport, no abnormalities were found on general physical and cardiovascular examinations of A1–A8. Four days after initial presentation (Day 66), puppy A8 was found dead after being observed and apparently healthy 2 h earlier.Diagnostic assessment included pathologic evaluation of animals who died. Diagnostic testing for living animals included PCR and ELISA testing for CPV-2, echocardiography, ECG, and cTnI.Postmortem examination at Wisconsin Veterinary Diagnostic Laboratory for Puppy A8 revealed cardiac dilatation with multifocal and locally extensive areas of myocardial pallor in the left and right ventricles . Cut surSignificant histologic findings commonly seen with heart failure were present in the lungs, liver and heart. The lungs displayed moderate alveolar histiocytosis. The liver had moderate centrilobular congestion and moderate lipid-type hepatocellular vacuolar change. The heart exhibited severe, chronic, multifocal and locally extensive myofiber atrophy, loss, and replacement with fibrous connective tissue. Low to moderate numbers of lymphoplasmacytic inflammatory cells infiltrated the myocardium.A section of the cardiac ventricular wall which exhibited pallor was strongly positive for CPV-2 on real time PCR with a 24.3 cycle threshold (Ct). Although no histologic changes were present in the intestines, samples from that tissue were strongly positive on real time PCR (Ct = 24.0) as well.Results of the necropsy findings supported PMC as a cause of death for A8.Samples were collected and tested by RT-PCR for each of the remaining puppies on Day 62 .One week later, on Day 70, on initial presentation at University of Wisconsin Veterinary Teaching Hospital, rectal swabs were collected from each puppy (A1–A7) for in house testing and submitted for a CPV-2 rapid ELISA antigen. Results are presented in The puppies (11 weeks old) were then presented to the University of Wisconsin Veterinary Cardiology service. Each puppy was given a brief exam and sedated with 0.5 mg/kg butorphanol intramuscularly to facilitate handling during cardiac diagnostics. Each puppy was auscultated right before injection. Echocardiography  was performed in a standard fashion. Echocardiographic measurements  were all within normal limits for puppies A1–A7, with results provided in Puppy A4 and puppy A6 both had hyperechoic areas in the left ventricle that could represent local ischemia at the time of their 11 week echocardiogram . EchocarStandard practices for ECG  were followed for patients A1–A7. A normal sinus rhythm was reported in A1–A7, no arrythmia, conduction abnormalities, nor were ST segment changes noted .Upon completion of cardiac evaluation on day 70, blood was taken from the jugular vein of each pup and submitted for cTnI testing to University of Wisconsin Health. The veterinary cardiologist was blinded to the cTnI results until the echocardiograms were evaluated and a formal report submitted.On day 103, a second sample was collected from the cephalic vein of each puppy and sent to University of Wisconsin Health for a recheck cTnI. Puppies A1 and A4 had a third sample taken for cTnI testing on day 640. As before, these samples were submitted to the University of Wisconsin Health. Results are listed in This case report presents 5 years of positive follow-up for seven surviving puppies in a litter affected by PMC which is three times longer than has been previously reported . PreviouThe variable outcomes of puppies in the litter described in this case report exemplify the importance of diagnostic necropsy in cases where sudden death affects a litter, particularly when exposure to parvovirus is suspected. Point of care testing was used three different times during this case report, and none of the fecal swab-based point of care tests (LFI and SNAP™ Parvo) could detect parvovirus infection in any of the puppies A1–A10. Even in A9 and A8, where each puppy had clear evidence of PMC on necropsy exam, as demonstrated by histological evidence of parvovirus infection in the heart (A9) or positive PCR results (A8), point of care fecal swab did not detect CPV-2. Previous reports demonstrate that infectious virus can be isolated from the hearts of puppies with PMC and that some puppies will only have infection of the myocardium rather than infection in both the heart and small intestine , 7. SincWhen fecal PCR testing was performed, positive tests with high Ct  values were reported for puppies A2 and A4–A7. The presence of a positive PCR test in this group of puppies may be related either to recent vaccination or infection with live virus but cannot be differentiated in this case , 20.Puppies affected by PMC described in previous reports fit roughly into two categories: (1) puppies who die acutely in the first weeks of life (~2–7 weeks), and (2) puppies and dogs who die months after birth up until the second year of life , 18, 21.In this case report, consistent with what has been described for this first category, one pup died at 45 days old. Histopathology showed infection of cardiomyocytes with viral inclusion bodies with low numbers of lymphocytes and plasma cells signifying infection of cardiomyocytes with parvovirus and the inflammatory response to viral infection . AnotherThis report also identifies and follows a third category of puppies. These rarely reported long-term survivors did not die suddenly in early puppyhood, nor did they experience the progressive heart disease that eventually causes heart failure or fatal arrythmias by age two. Because adoption outcomes were readily available for the puppies in this litter, ECG, echocardiography, and cTnI were utilized in hopes of prognosticating for the seven surviving puppies. These seven remaining puppies have gone on to live in their adoptive homes for 5 years.Studies and cases where echocardiographic information for dogs with PMC are rare because most of the cases were reported in the late 1970s and the 1980s when the use of echocardiography in dogs was not commonplace. In two contemporary reports, echocardiography was used to evaluate cardiac function in dogs with clinical illness from parvovirus enteritis. In both reports, dogs who ultimately did not survive had significant reduction in left ventricular function based on echocardiographic parameters consistent with DCM phenotype , 24. RepParvoviral myocarditis has been characterized by ECG since cases were first recognized in the late 1970s. Because ECG was a readily available diagnostic, it was frequently utilized throughout the study periods described but examples commonly included single puppies and, in some cases, ECGs reported were recorded just days before puppies died. These studies report on several abnormalities seen on ECG which may be relevant to prognostication for PMC including ST segment elevation, QRS abnormalities and arrythmias , 15, 18.S-T segment elevation has been associated with myocardial ischemia in dogs . But whiIn addition to conduction abnormalities, sinus tachycardia, ventricular tachycardia and premature ventricular complexes have been occasionally reported in puppies that were diagnosed with PMC , 15, 18.The seven puppies evaluated in this study did not demonstrate any of the ECG abnormalities described above . For theCardiac troponin I is one of three subunits in a larger complex that mediates the starting and stopping of contraction within each sarcomere of a cardiomyocyte and can be released into the bloodstream when cardiomyocytes are injured . CardiacThe use of cTnI is also documented in dogs with CPV-2. Bastan et al. demonstrated that puppies with naturally occurring CPV-2 enteritis who died from the disease and had evidence of myocarditis at necropsy had significantly higher cTnI values (cTnI >0.8 ng/ml) compared with the values in dogs who either survived or died but had no cardiac involvement at necropsy (cTnI < 0.156 ng/ml) . Two othIn this case report, cTnI for all seven puppies fell within the range for healthy dogs at both the initial test and at the 1 month recheck, and among individuals cTnI values changed very little between the initial and final values  Table 1Table 1. The COVID-19 pandemic interfered with planned recheck echocardiograms and cTnI values for all puppies, except A1 and A4. A verbal check-in regarding the fate of the rest of the litter was substituted for recheck diagnostics for A2–3 and A5–7.Within the litter of puppies described in this case report there were three different outcomes fitting with the three categories described: (1) one puppy died acutely at day 45 with evidence of parvovirus infection of cardiomyocytes (acute myocarditis), (2) another puppy died acutely at day 77 with histological evidence of extensive myocardial fibrosis and gross pathology evidence of DCM phenotype, and (3) the seven surviving puppies. The seven surviving puppies (A1–A7) have gone on to be sterilized and adopted with information about PMC. They are now nearly 5 years old. Two of the puppies were adopted even prior to the completion of all the initial diagnostic testing, indicating that even when prognosis is unclear, adopters who are provided with the available medical information regarding the puppies' health history were still willing to give these puppies a home. To the best of the authors' knowledge, this is the first time that the longer-term survival of puppies who came from a litter affected by PMC has been reported.It is unclear what causes the variation of outcomes within a group of puppies infected with parvovirus in the perinatal period, but based on the findings of this case report it appears that not all puppies in litters affected by PMC will have a poor prognosis. Demonstrating that puppies do not have compromised cardiac health using echocardiography, ECG, and cTnI may be a useful platform for identifying those puppies with a more optimistic prognosis in PMC affected litters.The original contributions presented in the study are included in the article/Written informed consent was obtained from the participant/patient(s) for the publication of this case report.BD: substantial contributions to the conception of the work, acquisition of samples, analysis, interpretation of data for the work, and drafted the manuscript. HK: contributions to the conception of the work, acquisition, analysis, and interpretation of data for the work. CA: interpreted the data, drafted the manuscript, and coordinated communication among the authors. AL and PB: provided pathology descriptions, analysis, and as well as pathology figures. SN: advised on the concept of the work, interpreted data, and drafted the manuscript. All authors critically reviewed the manuscript, provided revisions, and approved the submitted manuscript.


Background and Objectives: This study evaluated the effectiveness of Hyalofast cartilage repair surgery with an early, full load-bearing rehabilitation program one day after the operation for reducing the time needed for professional athletes to return to play. Materials and Methods: This prospective study included 49 patients aged between 19 and 38 years who had undergone surgical reconstruction of cartilage using the microfracture technique combined with a Hyalofast scaffold. All patients were active professional athletes. Early rehabilitation was implemented from the first postoperative day, fully loading the operated limb. A clinical evaluation was based on the KOOS and SF-36 questionnaires used during subsequent follow-up visits. All patients underwent magnetic resonance imaging (MRI) to evaluate the effect of the surgery after one year. Results: The clinical results demonstrated a statistically significant improvement in the number of complaints about pain and in the quality of life of the patients, measured in all of the applied scales, with comparisons made between six months or one year post-surgery and pre-surgery. Importantly for athletes, the parameter related to sports and recreation improved from 14 ± 11.1 to 95 ± 7.7 6 months after surgery and to 99.8 ± 1.8 one year after surgery. The overall quality of life score improved from 30 ± 18 to 88 ± 8.8 one year after surgery. Conclusions: These results show that this approach significantly shortened the time needed for the athletes to return to sports at the same level as before the surgery (athletes returned to sports in approximately 2.5–3 months). The mean follow-up time was 19.75 months. This technique can be considered a viable option for the treatment of cartilage injuries in professional athletes, allowing them to return to play more quickly in a safe and healthy way. The treatment of articular cartilage injuries has been a challenge for doctors around the world for decades. This is due to the fact that articular cartilage has limited healing potential due to a lack of blood vessels and nerve connections. Tissue nutrition depends mainly on the diffusion of nutrients from the joint cavity ,2,3,4. ACurrent therapeutic solutions are aimed at reducing cartilage load and promoting the cartilage regeneration process ,20,21,22Bone marrow stimulation techniques (such as microfracturing) have been used since the 1950s, and were originally developed by Pridie and Steadman when they tried to induce subchondral bone bleeding to release multipotent mesenchymal stem cells and growth factors into the defect site of cartilage, leading to cartilage repair ,42. The TM membrane—a fibrin substrate that stimulates the cartilage to grow—has proven to be an interesting alternative for patients with intensive joint pain [The repair of cartilage defects with a Hyalofastint pain ,47,48,49int pain ,51.However, it should be noted that despite significant advances in the treatment of cartilage damage, recovery can be prolonged, significantly delaying the return to sports for athletes. Therefore, the preparation of an appropriate rehabilitation program is essential to optimize the results of the surgical treatment of cartilage injuries. Rehabilitation protocols can significantly improve the cartilage repair process, thereby increasing athletes’ physical activity and reducing the risk of re-injury . This isThe objective of this study was to investigate the effectiveness of arthroscopic surgery using the microfracture technique combined with a Hyalofast scaffold to treat knee cartilage injuries followed by an intensive rehabilitation program with full load bearing just after surgery among professional athletes. This article highlights the novelty of a full weight-bearing approach for the treated joint very shortly after cartilage reconstruction surgery, along with an intensive rehabilitation program. This approach is supported by MRI evidence, and the KOOS and SF-36 questionnaire results suggesting that early resumption of a full range of motion can improve cartilage healing. It also emphasizes the importance of individualized rehabilitation protocols that consider the patient’s specific condition and progress, as well as close monitoring by a healthcare professional. Compared to the literature data on rehabilitation programs, the proposed treatment approach allows patients to have full range of motion with full weight bearing without the use of orthoses as early as one day after surgery (under continuous monitoring by attending physicians). The combination of full weight bearing and intensive rehabilitation may offer a promising approach to enhance postoperative recovery and optimize the required recovery time for athletes undergoing cartilage reconstruction surgery (reducing the required time needed to return to sports at pre-injury level to 2.5–3 months).The patients were classified for the procedure according to the ICRS classification. In total, 49 patients, 34 men and 15 women aged 19–38 years (mean = 30 years), underwent cartilage reconstruction surgery. All of these patients were active professional athletes  . Twenty-The inclusion criteria of the study included active athletes with grade IV articular cartilage focal damage who were indicated for surgery and who did not have a history of cartilage surgery on the affected knee joint. The exclusion criteria included patients under 18 years old or above 50 years old, patients with muscle disorders such as myasthenia gravis, progressive malnutrition, or periodic paralysis. Patients who were not willing to cooperate with the treatment, as well as those who refused to rehabilitate under supervision at our clinic, were also excluded from this study. It was also impossible to include patients who had abnormal bleeding or an abnormal coagulation function and patients with incomplete follow-up and imaging data or a follow-up time of less than six months.The patients underwent cartilage regeneration surgery with the use of a Hyalofast membrane , microfractures, and tissue glue stabilization. After an MRI evaluation of the lesion size and location, a physical examination, an assessment of pain intensity, and verification of the failure of conservative treatment, the patients were referred for surgery by their attending physician. The study protocol was approved by the local ethics committee of the authors’ affiliated institution. All patients gave their written informed consent to participate in the study.The same team and surgeon performed all activities under general anesthesia. All procedures involving the knee joint were performed arthroscopically. The first step of surgery involved the insertion of the two classic anteromedial and anterolateral portals and an accurate assessment of the joint structures—ligaments, menisci, and type and depth of cartilage damage. In those patients with meniscal tears requiring removal, a partial or total meniscectomy was performed. After a careful cleaning of the lesion with the complete removal of damaged tissue, the size of the cartilage defect was assessed. Then, microfracturing was performed at the base of the defect using a dedicated set of tools A. FollowWe evaluated the treatment results comparing the patients’ preoperative situation with a functional assessment performed during the follow-up visits. The patients completed the SF-36 and KOOS questionnaires before surgery and at 2, 6, 12, and 24 weeks and 1 year after surgery. The Knee Osteoarthritis Outcome Score (KOOS) is composed of five subscales that assess pain severity (PAIN), symptoms (SYMPTOMS), function in daily living (ADL), function in sports and recreational activities (SPORT/REC), and quality of life (QOL) . The finNo brace was used, nor was any stabilization. After the operation, oedema and pain were controlled by a Game Ready cooling system and compression stockings  and a regeneration and massage system ). A physiotherapy program was started on the first day after the operation, including walking on the operated limb with a full load, supported by elbow crutches. The crutches were put away seven days after the operation. The patients were not limited in their range of movement after surgery. The physiotherapy protocol included resistance training, eccentric–concentric exercises, and exercises with full body weight, with a focus on force and increasing the movement range of the erector and flexor muscles of the knee  and HydroWorx underwater treadmills . The patients learned to carry out normal movement patterns under supervision. A strong emphasis was constantly placed on proprioception exercises under unstable ground conditions. The exercise package was completed by the manual activities of a physiotherapist. By three weeks after the surgery, the athletes had already started preliminary training sessions under the supervision of a physiotherapist and a physician. Additionally, 02 Prime Hyperbaric Chambers  and athletic compression sportswear  were also used during rehabilitation program. The rehabilitation room undergoes regular sanitary-epidemiological inspections. Due to its nature as a medical centre, each piece of equipment has a current mandatory technical passport that is renewed once a year. Each patient is guided by the same therapist throughout the entire treatment process, and the same person operates the rehabilitation equipment.t-test or a Mann–Whitney U-test for continuous data, while for categorical data, a Pearson’s chi-square test was used. A p-value of less than 0.05 was considered significant.The obtained results are presented as the mean value ±standard deviation (SD). All statistical data were analyzed using STATA software . Differences were compared using an unpaired The patient evaluation results from two questionnaires (KOOS and SF-36) are presented below. The obtained results from the KOOS and SF-36 questionnaires show a significant increase in the patients’ health status and quality of life as soon as six months after surgery. On the KOOS scale, pain improved from 55 ± 18.2 to 96 ± 4.2 by the sixth month and to 99.5 ± 2.3 by the 12th month. The symptoms improved from 46 ± 16.6 to 93 ± 6.5 by the sixth month and to 99.5 ± 2.3 by the 12th month. The parameter for activities of daily living increased from 57 ± 25.5 to 100 ± 0.0 one year after surgery. Importantly for athletes, the parameter related to sports and recreation improved from 14 ± 11.1 to 95 ± 7.7 by six months after surgery and to 99.8 ± 1.8 one year after surgery. The overall quality of life score improved from 30 ± 18 to 88 ± 8.8 one year after surgery. There was a statistically significant difference in the KOOS scores evaluated before and at 6 and 12 months after surgery. A statistically significant difference was noted in the following SF-36 subscale scores at six months postoperatively: bodily pain improved from 36 ± 18.7 to 78 ± 15.6 and physical function from 34 ± 18.5 to 99 ± 2.0. One year after surgery, a statistically significant difference was noted for all SF-36 parameters.The results from a comparison of the KOOS and SF-36 scores for different sports indicate the best progress for the basketball and football players . For mosAn additional analysis was performed to determine the difference between those patients who underwent additional treatment of their meniscal lesions during the treatment of their cartilage injuries and those patients treated solely for chondral changes . The SF-Overall, three to four weeks after the surgery, all of the patients returned to activities of daily living without pain symptoms. The time of return to sports varied and ranged from two to four months. All professional athletes (100%) returned to practicing their sporting discipline at their pre-injury level (mean time = 2.5–3 months). At the 6- and 12-month follow-up visits, we did not observe any abnormalities in terms of range of motion, oedema, or pain in the treated joint.All patients underwent a postoperative SIGNA 1.5T HDx MRI scan. Similar results were obtained for all groups of athletes included in this study. An example of the results is shown in No complications were found after surgery, such as infection, bleeding, liver/kidney dysfunction, or other knee injury.Several techniques for the surgical treatment of cartilage defects have been developed in recent years. The choice of the most appropriate method is still controversial. Arthroplasty is one of the most common solutions described in the literature and used in clinical practice for the treatment of high-grade chondral defects . It is aThe use of the Hyalofast scaffold has showed positive results in other research. Quiceno conducted a study to evaluate the short- and medium-term outcomes of patients treated with autologous matrix-induced chondrogenesis (AMIC) using a hyaluronic acid-based scaffold with grade IV cartilage changes in the knee. The results of this study indicated that patients who underwent this procedure demonstrated satisfactory results throughout the observation period . In GobbThe data available in the literature demonstrate that use of the microfracture technique to improve the function of affected joints ,67 is asAs the problem of cartilage damage affects many professional athletes, physicians are faced with the duration of the recovery period and with the possibility of regaining the pre-injury performance level. Della Villa et al., in their study, suggested the need to delay the moment when the treated joint can be fully loaded . They juIn a systematic review published in 2009, Mithoefer et al. assessed the recovery time and the percentage of professional athletes returning to active sports after articular cartilage damage . A returOur treatment option is unique in comparison to the published literature. Some other authors have suggested that weight bearing on the limb should be restricted for a period of four, six, and even eight weeks and indicate the need for a range of motion (ROM)-limiting orthosis. In comparison to this approach, early mobilization and early full weight bearing of the treated joint (the first day after surgery), with the use of crutches being stopped two weeks later, seems to create quite a radical approach. Such a procedure, as evidenced by our paper, not only does not impact the cartilage regeneration process adversely, but it enables the patient to rapidly regain sporting activity and independence. In general, one to two weeks after surgery the patient can function in their daily life without pain. In all patients presented, elimination of pain was sustained with no recurrence. Three weeks after surgery, the patients started a pitch training program adapted to the sporting discipline practiced. This is a crucial element in preparing the patient to return to sports and to team training, which we achieved by preventing patients from developing a fear of re-injury and by increasing their confidence in the use of the treated limb. The longest follow-up period was 24 months. During that period, we did not observe any treatment failure or recurrence of symptoms within the treated knee joint. Moreover, MRI scans showed, in all cases, the healing of the defects with regenerated cartilage and elimination of the periosteal reaction.The limitations of our study include not having a control sample treated with a less-intensive rehabilitation program. However, in reality, patients who are not active athletes usually do not show such motivation during the rehabilitation period. Additionally, conducting a randomized trial with a longer follow-up period would provide more observations, but such a study may be difficult to conduct.In this work, the effectiveness of knee cartilage defect treatment was analyzed. Hyalofast membranes, combined with microfracture surgery and a tissue adhesive, supported by an intensive postoperative physiotherapy protocol, significantly improved the quality and comfort of life of the operated patients. Compared to the preoperative and follow-up period, the proposed treatment procedure allows a significant reduction in the time needed for the patient to return to normal and sporting activities, showing excellent results for athletes. The implementation of an intensive postoperative rehabilitation program is key to optimizing the results of cartilage defect repairs and influences the time needed to return to full athletic performance. Based on the conclusions of the present study, there are several areas for future research on knee cartilage defect treatment. Firstly, additional randomized trials could be conducted to confirm the findings of the current study and to assess the long-term outcomes of the proposed treatment procedure. Further investigation could also examine the effectiveness of the proposed treatment procedure in different patient populations, such as older individuals or those with more severe cartilage damage. Secondly, future research could focus on identifying the optimal international postoperative rehabilitation protocol for patients undergoing knee cartilage defect treatment and also other injuries . This could involve exploring different exercise programs or evaluating the use of other therapeutic modalities, such as those proposed in this article.


Replacement was conducted by using a virus neutralisation test on cell cultures prior to infecting and killing the animals. We determined that the antibodies produced by the tested vaccines did not have sufficient neutralising properties, and the project was terminated. Approximately half of the golden hamsters that were no longer needed in the procedures were rehomed and we received very encouraging feedback from adopters.In 2020, Slovenia joined the global effort to develop effective vaccines and drugs to treat COVID-19. Two vaccine candidates developed in previous studies were selected and tested in the golden hamster model using four different vaccination protocols. We followed the required 3Rs principle when performing the procedures on the animals: we Effective vaccines are needed to fight the COVID-19 pandemic. Forty golden hamsters were inoculated with two promising vaccine candidates and eighteen animals were used in pilot trials with viral challenge. ELISA assays were performed to determine endpoint serum titres for specific antibodies and virus neutralisation tests were used to evaluate the efficacy of antibodies. All tests with serum from vaccinated hamsters were negative even after booster vaccinations and changes in vaccination protocol. We concluded that antibodies did not have sufficient neutralising properties. Refinements were observed at all steps, and the in vitro method  presented a replacement measure and ultimately lead to a reduction in the total number of animals used in the project. The institutional animal welfare officer and institutional designated veterinarian approved the reuse or rehoming of the surplus animals. Simple socialization procedures were performed and ultimately 19 animals were rehomed, and feedback was collected. Recently, FELASA published recommendations for rehoming of animals used for scientific and educational purposes, with species-specific guidelines, including mice, rats, and rabbits. Based on our positive experience and feedback from adopters, we concluded that the rehoming of rodents, including hamsters, is not only possible, but highly recommended. Cricetinae—show this homology, and project proposals using hamsters have nearly tripled in Germany, for example, compared with pre-pandemic years [Mesocriccetus auratus), is the most common in publications, although it has been shown that the Chinese hamster (Cricetulus griseus) actually develops more severe signs of disease [Phodopus roborovskii) can sometimes be fatal [In 2020, researchers in Slovenia joined a global race to find effective therapies and vaccines against the COVID-19 pandemic. In this context, small animal models are essential, as preclinical studies in animals are crucial for basic and applied research in most infection studies. Several SARS-CoV-2 animal models based on ACE2 receptor homology have been studied, including nonhuman primates, transgenic mice, ferrets, cats, and hamsters [ disease , infectibe fatal , and botbe fatal , served be fatal . They arVaccine efficacy (and safety) testing has been characterized by extensive use of laboratory animals. Working groups have been formed and recommendations made to develop the required 3R—Replacement, Reduction, and Refinement—methods that include a nonclinical endpoint, ultimately reducing the number of animals and decreasing severity levels of procedures on animals (refinement) . DetermiIn the present study, we tested two promising vaccine candidates based on previous research  on an apMesocricetus auratus) of strain HsdHan AURA were supplied by Envigo  from a UK-based breeding stock  and 18 animals  were supplied by Janvier Laboratories  of strain RjHan: AURA. Both outbred lines originate from Zentralinstitut fur Versuchstiere, Hannover, Germany. The animals were 7–8 weeks old at purchase and housed in same-sex pairs in conventional polypropylene cages with filter covers (Techniplast Eurostandard type III). Between week 8 and 10, all females and some males started to fight despite increased enrichment. Between the 10th and 12th week, we separated all animals to prevent fighting and they remained in single housing until the end of the vaccination project. A total of 40 were used for vaccine testing: 32 with the first vaccine candidate and 8 animals with another vaccine candidate. The animals were kept in dedicated rodent rooms at the Faculty of Veterinary Medicine, University of Ljubljana, which provided a controlled environment with a relative humidity of 45–60%, a temperature between 21 and 23 °C, and a 12:12 light—dark cycle. Feed and water were given ad libitum; pelleted irradiated feed contained 21% crude protein (Sniff diets S8189-S098). Irradiated wood fibre bedding was used ; for enrichment, sterilized paper strip nesting material , sterilized aspen wood gnawing blocks , and cardboard or polypropylene tubes for hiding were offered. Golden hamsters  using an animal biocontainment system (Techniplast IsoRat900 N with Teklad Isoplast bedding)—designated A-BSL3 . For the second and third trials, animals were 8 weeks old and kept in pairs or threes as no territorial aggression occurred yet. In total, 58 animals were used for the project. All animal procedures were performed in accordance with the EU Directive (2010/63/EU), approved by the Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection  and its Ethical Committee (Decisions U34401-18/2020/8 and U34401-11/2021/9) and reports were prepared according to PREPARE and ARRIVE guidelines.Two different routes of administration and two concentrations of the best vaccine candidate identified from previous in vitro and mouse immune response tests  were selFor the final vaccination protocol, 8 naive animals (7 months old) were used and vaccinated with recombinant RBD protein and Complete Freund’s adjuvant  in a 1:1 ratio, 50 μL subcutaneously (s.c.) in two skin folds—just caudal to the elbow joint (retroaxillary) and cranial to the stifle .® 500 Serum Gel, Sarstedt, Germany), allowed to coagulate for 8 h or overnight in the refrigerator, and centrifuged at 9000× g for 10 min. The separated serum was frozen at −22 °C until analysis.Blood samples were collected from the animals immediately before each vaccination and 2 and 4 weeks after the last booster vaccination, always under general anaesthesia (procedure described in Refinements down the page). Blood was transferred to a mini-tube containing a gel clot activator . Half of the food pellets were always offered on the floor of the cage to allow for hamster-specific storage behaviour. Blood samples were collected under general anaesthesia at the junction of the cranial vena cava, external jugular vein, and subclavian vein. Isoflurane was used as 4.5% in the induction chamber, maintained by 2.5–3.5% via the face mask: the animal was placed in dorsal recumbency, the puncture site was located on the left or right craniolateral to the manubrium sterni, and the needle was inserted in a caudal direction toward the contralateral hip joint . The punRandomization was performed during acclimation of the animals—one animal caretaker randomly assigned animals to cages in pairs; when pairs were separated, cages and animals were renumbered by the second caretaker. Ear notches for individual ID were made under general anaesthesia at the first blood draw. The allocation of the first vaccination protocol was conducted by the researcher, who did not know the allocation of each animal ID, except for the sex of the animals. The remaining vaccinations were allocated based on preliminary results. The researchers who performed titration, ELISA, PCR, VNT tests, or histology were blinded during the analysis.ELISA was performed to determine endpoint titres for designated specific antibodies as described before . Sera frTo test the general neutralisation properties of the antibodies detected in hamster sera, a pseudovirus (PV) neutralisation assay was carried out as described before . DetailsSerum samples were tested for the presence of neutralising SARS-CoV-2 antibodies using a virus neutralisation test (VNT). The SARS-CoV-2 strain Slovenia/SI-4265/20 was provided by the European Virus Archive and the Vero E6 cell line (African green monkey kidney cells) was provided by ATCC: VERO C1008; CRL-1586 [4 TCID 50/mL diluted in Eagle’s Minimum Essential Medium (EMEM), observed daily for clinical signs, and killed with CO2 and exsanguination on days 2 , 4 , and 7  after inoculation to determine the best time point and tissue samples. Nasal conchae, trachea, lung tissue, duodenum, and whole brain were collected. FLOQSwabs  from the caudal nasal cavity, trachea, lung’s cutting surface, and duodenum were also taken for RT-qPCR. Based on the initial results, a second trial with a group of 6 animals  was conducted. The animals were inoculated intranasally (i.n.) with 50 μL (25 μL per nostril) of the virus with a titre of 5 × 104 TCID50/mL, monitored daily for clinical signs, and killed with exsanguination (cardiocentesis) under inhalation anaesthesia on the fourth day after inoculation. Because clinical and pathologic signs were low, we increased the viral load and tested on an additional 6 animals —inoculated i.n. with 50 μL (25 μL per nostril) of the virus with a titre of 1 × 106 TCID50/mL—designated as the third pilot trial. Animals were monitored and weighed every 24 h and killed by exsanguination under inhalation anaesthesia on day 4. Blood and lung tissue samples were collected—the entire left lung lobe was placed in 4% buffered paraformaldehyde for histological processing, and the entire right lung was weighed and homogenized in sterile Eagle’s Minimum Essential Medium (EMEM). The clinical score sheet was used for signs of lethargy, ruffled coat, hunched posture, laboured breathing, nasal discharge, cyanosis, and facial grimace: 1 for mild and 2 for severe manifestation . The same trained person observed the animals and filled in the evaluation forms. She was not blind to the study, but she was blind to animal ID each time to start with as the cage position might have changed at the first morning inspection by the animal caretaker. Animals in A-BSL3 were identified by cage cards and abdominal marking (blue pen) to separate between 2 or 3 animals in the cage only upon close inspection.In the first pilot trial, 6 naïve hamsters  were intranasally inoculated with 25 μL of the SARS-CoV-2 virus strain Slovenia/SI-4265/20 (European Virus Archive) with a titre of 5 × 1050/mL) [Virus titres were determined by titration of sample homogenate suspensions on Vero E6 cells and measured as the 50% infectious tissue culture dose (TCID50/mL) ,17,18. SThe swabs of organs were individually vortexed in 2 mL phosphate-buffered saline for 2 min prior to genomic nucleic acid extraction. Total RNA and DNA were extracted from 140 µL of sample supernatant by the QIAamp Viral RNA Mini Kit  according to the manufacturer’s instructions. Viral RNA was detected with the real-time assay RT-qPCR targeting the E gene of SARS-CoV-2 using the primers and probe described by Corman et al. . RT-qPCRThe left lung was fixed in 4% buffered paraformaldehyde for 3–4 days, embedded in paraffin, and cut into 5 µm horizontal sections and stained either with HE or the location of the virus with SARS-CoV-1/2 Spike Protein (2B3E5) mouse mAb  was labelled. More details on this procedure can be found in the The prospect of reusing or rehoming the animals was not included in the project proposal, as all animals were expected to enter the A-BSL3 facility for viral challenge and be killed there. Based on the results during the vaccination process, it was decided to omit the viral challenge procedure on vaccinated animals because antibodies produced did not have (detectable) neutralising properties and infecting the animals would not bring any new data needed for the purpose of the project. The facility animal welfare officer was consulted to approve the reuse or rehoming of the 40 surplus animals—the animals that were vaccinated but not exposed to the virus. The institutional designated veterinarian certified that most of the animals were in good health and posed no threat to the environment or human health, so they could be reused or rehomed, even though they were about halfway through their life expectancy at that point: 16 months old and life expectancy is between 2 and 3 years. A total of 16 animals in suboptimal condition were sacrificed for tissue samples for basic research and controls. In the meantime, simple socialization measures were implemented, primarily through giving fresh food treats, frequent handling, and alternative environmental enrichment. Another 5 animals that did not adapt well to handling were deemed unsuitable for rehoming as pets. Adopters were initially institutional and later external. Prospective adopters contacted the person responsible for the hamsters, an interview was conducted to confirm the adopter’s knowledge of hamster care, a simple adoption contract was signed, and voluntary follow-up meetings were held over the following months. In the adoption contract, there was also basic information on procedures the animals were subjected to and approvals of the designated veterinarian and welfare officer. DNA vaccines to date do not persist or even biodistribute throughout the body of the vaccine recipient when delivered parenterally into muscle, subcutaneous tissue, or dermal layers. The local response to plasmid DNA inoculation is that cells take up the plasmid and then express the immunogen(s)-encoded mechanisms; the nucleic acid is degraded by normal molecular mechanisms. As a consequence, the plasmid DNA clears from the injection site over time . A minimwww.enka.si. The link to the questionnaire was sent to 17 adopters, and 11 responded. The (translated) questionnaire is added in the Out of 40 animals, 19 animals were designated fit for rehoming and all found new homes. Eighteen months after the last adoption, a simple anonymous online survey was conducted via a local popular webpage, Sixty hamsters were planned to test the plasmid DNA RBD-bann vaccine  as well as the protein-based RBD-bann. The supplier was only able to supply forty hamsters, which we divided into four groups for the plasmid DNA vaccine and one group for the protein-based vaccine, with each group consisting of four male and four female hamsters. Prior to vaccination, the hamsters tested negative for SARS-CoV-2  and a blood sample was taken. This was used as a control—all antibody titres were compared with a control serum from each individual. Protein RBD bann was tested on 8 naïve hamsters after the initial ELISA and VNT results and also on 10 animals in the form of booster doses after two doses of the DNA vaccine. The diagram in With 18 animals from the new supplier, we conducted pilot tests with the virus in an animal biosafety level 3 (A-BSL3) laboratory. None of the vaccinated animals entered A-BSL3 and were not infected with the virus.As expected, only the booster dose resulted in high levels of IgG titres in the hamster serum presented in the graph in Neutralisation of the pseudovirus showed little effect, and insignificant lower or higher luciferase signal levels did not correspond to group assignment .No neutralising antibodies were detected by VNT in any of the hamster serum samples tested; all dilutions resulted in cytopathogenic effect CPE—. The pos4 TCID50/mL SARS-CoV-2 strain Slovenia/SI-4265/20, day 4 after inoculation was selected as the best time for the clinical endpoint, and swabs from the caudal nasal cavity were more representative than swabs from the oropharynx but were time-consuming to obtain in an A-BSL3 facility. In the duodenum and trachea, viral RNA was either not detected or was very low  in the third trial, we observed more obvious clinical signs: lethargy and ruffled coat, and 50% of animals had a mild nasal discharge and showed laboured breathing. The amount of virus in the lung tissue detected by viral titre from lung homogenate increased, viral RNA RT-qPCR Ct values dropped, and the ΔCt between the second and third trial were significantly different at p = 0.002 as presented in the To determine the best time for euthanasia, tissue, and method of sampling, pilot experiments were conducted with a minimal number of animals. Non-vaccinated animals were infected with 25 µL 5 × 10d weight . By incrThe use of 6 extra animals  or pilot trials as a whole  might contradict the reduction principle of 3R but was considered as refinement as the virulence of specific SARS-CoV-2 strains for golden hamsters was not known. None of the vaccinated animals were subjected to the viral challenge procedure.Of the 40 animals vaccinated, 1 developed otitis media, was treated at the Faculty’s Clinic for birds, small mammals, and reptiles, and was eventually adopted by the attending veterinarian with the approval of the institutional animal welfare officer. The designated veterinarian performed the clinical examination of the remaining animals. Three were in worse condition, likely due to their advanced age (over 1 year old), and were euthanized. Another 12 animals were selected for reuse for scientific or educational purposes, humanely killed, and tissue samples collected. Socialization measures were taken for the remaining 24 animals. Five of the animals did not respond well to frequent handling, usually hiding in the tunnel when staff were present, so they were not candidates for rehoming as pets. A total of 19 hamsters were eventually adopted; the first six adopters were internal, the next seven semi-internal (veterinary students and trainees in the LAS course of October 2021), and the rest external. The word was spread through friends and relatives. Internal adopters provided frequent feedback, and a year and a half later, a general opinion was obtained through an anonymous online survey , must not only be due to the  low inoculum dose  but are The immunohistochemistry score in our pilot control group was weak and only slightly improved after increasing the inoculation dose. Most of the positive signals were located in the bronchi and only sparse interstitial locations in two individual animals. It should be noted that pathologic changes are not evenly distributed and, therefore, a wide transverse section of the entire lung lobe, ideally the left non-lobulated one, is needed . At leasResearchers always strive to minimize the number of animals used as long as a relevant statistical analysis can be performed. When working with dangerous infectious agents, downsizing animal numbers may even be enforced due to the limited capacity of A-BSL3 facilities. The risk of over-reduction exists and thus challenges the reliability and reproducibility of the studies—the factors that are often referred to “Rs beyond the 3R”. For our study, we managed to organise a capacity to house up to 60 hamsters simultaneously in a renovated A-BSL3 but were unexpectedly faced with limited availability of the species. Instead of the planned 60 hamsters for different vaccination protocols and three control groups with placebo injections, we could initially acquire only 40 animals. A decision was made to optimise the experiment design: test all the planned protocols, but for the controls/blanks, use the sera of the same hamsters before the vaccination. Such a reduction principle—comparing responses at later time points to a baseline level—is commonly used in toxicology . We did Regarding the reliability of the studies, we need to highlight the sex differences, as we have observed more pronounced clinical symptoms in males and this has subsequently been well documented ,38. StudWe found no reports of negative results that would explain the lack of neutralising properties of hamster antibodies, and overall, not that many papers describing vaccine testing in golden hamsters were found. It is likely that most laboratories started using genetically modified mice (hACE2 mice and further variants) when they became more readily available, because after the initial boom, the number of studies using hamsters declined slightly . Withhol2. With vaccination protocols, animals were kept longer in the conventional facility, and soon after reaching sexual maturity, all females began to fight, inflicting bite wounds. To create comparable conditions, we separated both sexes and switched to single housing in these procedures.Refinements such as environmental enrichments, friendly handling, and general anaesthesia for all inoculations and blood sampling have been introduced for the general welfare of the animals, even though minimal pain procedures were unlikely to affect the final immunological outcome. We hypothesize that minimizing the pain and other negative associations with humans contributed to rapid and smooth socialization in most animals, as confirmed by adopter feedback. It is important to note that this species exhibits burrowing behaviour , and theThe final refinement represented the decision to rehome some of the animals. The rehoming process was initially met with some scepticism by the research team but was then supported by the animal welfare officer, approved by the designated veterinarian, and accepted with great satisfaction in the end. Most adopters were overjoyed to give the animal a second chance in life and would gladly adopt a similar animal next time. We do not know the opinion of the adopters who did not respond to the survey (35%)—their experience may be different but would not be the deciding factor in this case. To date, there are no specific institutional  guidelines or protocols regarding rehoming animals used or bred for research purposes. In Slovenia up until now, only farm and wild animals that have undergone non-invasive or minimal harm procedures have been considered suitable for “return to herd” or “set free” in the case of wildlife. Once the ice was broken, future project proposals may consider rehoming laboratory animals at this and other institutions as public interest—including for rodents—appears to be greater than scientists would generally hope for. Success stories, including rehoming rodents, come from other European countries. Some have chosen to work with animal welfare NGOs  . This isAs laboratory animal science faces significant challenges, openness and transparency with the public is now encouraged. Citing MacArthur Clark , “A balaRefinement is a ubiquitous tool in almost every step of research projects involving procedures on animals. Pilot trials are highly recommended when new techniques, equipment, infectious agents, etc., are used because it confirms the observation of the reduction principle. In our pilot viral challenge test, we found that we needed to use a higher viral load of a particular virus strain to elicit a sufficient clinical response as originally planned—and this would prevent the need to repeat the entire experiment with vaccinated animals. The virus neutralisation test is an example of an in vitro prerequisite that may reduce the severity or even total number of animals in the project. Because it could not demonstrate the neutralising properties of the hamster antibodies on vaccine candidates tested in our project, it served as a replacement for the in vivo viral challenge. The surviving golden hamsters were successfully rehomed in our study. Positive attitudes among both scientists and adopters of retired or surplus laboratory animals can improve public attitudes toward the use of animals for scientific purposes and help restore general confidence in science.