Saturated salt solution: a further step to a formaldehyde-free embalming method for veterinary gross anatomy.

In the field of veterinary anatomy, most of the specimens used in practical sessions are perfused with fixatives. Thus, they can be used for a longer time, reducing the number of animals for educational purposes. Formalin is the most commonly used fixative, consisting of a 37% formaldehyde solution. However, formaldehyde is a powerful irritant of the eyes and airways and is considered carcinogenic, causing nasopharyngeal cancer in exposed workers and professionals. In the present study, we explored an alternative method to avoid the use of formaldehyde in specimens used for gross anatomy practical sessions. We propose an inexpensive, non-toxic fixative that is available worldwide, such as sea salt. This method consists of a continuous perfusion of saturated salt solution for a period of 6-8 h, enabling drainage of the solution to avoid a weight increase of the specimen, and allowing salt to be retained in the tissue. The method is based on recirculation of the saturated salt solution instead of maceration. Perfused specimens retained their natural consistency and joint mobility, with no blood, resembling a piece of meat from the slaughterhouse. They could be used immediately without a maceration period, or stored in the fridge until use and then kept in a bath of saturated salt solution for future conservation. In the case of the former, no refrigeration was needed. The specimens did not have an irritating or offensive smell, and could be used for long sessions (several hours per day) and stored for long periods. However, the blood vessels used for perfusion determine the results: a less invasive approach (through common carotid arteries) gave good preservation of the musculoskeletal system, whereas more invasive access to cannulate the abdominal aorta and vena cava caudalis was required to achieve better preservation of the viscera. In conclusion, we propose that perfusion followed by immersion in a saturated salt solution is a good alternative method for the preservation of specimens used in the practical teaching of gross veterinary anatomy. It is a very simple and inexpensive technique, and is much healthier for users than traditional formalin. Moreover, specimens can be preserved for prolonged periods, and maintain a similar appearance and consistency to fresh material.

Benign acute childhood myositis: A 5-year retrospective study.

BACKGROUND: Benign acute childhood myositis (BACM) is a rare, transient, and self-limited inflammatory condition of the skeletal muscle that usually occurs after a viral infection. It affects pre-school and school-aged children at a median age of 6-9 years, with a male predominance. Patients can be managed with analgesia, rest, and adequate hydration at home. METHODS: A retrospective review was carried out of patients' clinical records with an elevated level of creatine phosphokinase (CPK) who presented to the pediatric emergency department between January 1, 2015 and December 31, 2019. RESULTS: Out of 174 cases of elevated CPK values, 100 corresponded to BACM-compatible clinical presentations (n = 96). There was a male predominance (77%) with a median age of 6 years. There were more cases registered in 2019 compared with the previous years. The most frequently reported prodromal symptoms were fever, cough, and rhinorrhea. Bilateral calf pain was the most frequently reported BACM symptom, followed by gait complaints and refusal to walk. Hospitalization was an independent predictor of CPK levels. In two cases there was myoglobinuria. The most common hematological findings were leukopenia and thrombocytopenia. CONCLUSION: Our results regarding male predominance, median age, and hematological findings are similar to the published reports. The outbreak during 2019 coincided with the 2019/2020 flu season. During this period, influenza B was the most prevalent virus and it is known to be the most myotrophic one. Physicians often take into consideration CPK levels during inpatient admission. All patients recovered fully, emphasizing the fact that BACM is benign, self-limited, and with an excellent prognosis.

Adult rabbits acquire resistance to lethal calicivirus infection by adoptive transfer of sera from infected young rabbits.

Calicivirus infection of adult rabbits induces the so-called rabbit haemorrhagic disease (RHD) that kills 90% or more of the infected animals; in contrast, young rabbits (up to 8-week-old animals) are resistant to the same infectious agent. We report that calicivirus inoculation of young rabbits induced moderate titres of antiviral antibodies. When these rabbits reached adulthood, a second calicivirus inoculation resulted in resistance to RHD and boosting of antibody titres in half of the rabbits. Adoptive transfer of sera from calicivirus-infected young rabbits to naïve adult rabbits conferred resistance to RHD. We conclude that calicivirus infection of young rabbits induces specific anti-calicivirus antibodies that will protect them from RHD when they reach adulthood.

Liver disease in young rabbits infected by calicivirus through nasal and oral routes.

Calicivirus infection causes rabbit haemorrhagic disease (RHD) that kills more than 90% of adult animals, whereas young rabbits are naturally resistant to this viral disease. It has been proposed that the different response of adult and young rabbits to calicivirus infection is due to absence of viral receptors in respiratory and digestive systems of young animals. We have searched for liver disease in 4-week-old rabbits inoculated with a calicivirus suspension by intranasal and oral routes. These young rabbits showed cell damage and mononuclear infiltration of the liver. The hepatic lesions were associated with mild to moderate increase in circulating transaminases. We conclude that the previously reported reduction of viral receptors in the epithelium of respiratory and digestive systems of young rabbits does not inhibit calicivirus from inducing liver disease in these hosts.

Severe leukopenia and liver biochemistry changes in adult rabbits after calicivirus infection.

Calicivirus infection is the major cause of the severe decrease in the stocks of wild and farm rabbits that has occurred worldwide during the last two decades. Adult rabbits (10-weeks-old) were experimentally infected with a calicivirus inoculum that killed all animals by causing rabbit haemorrhagic disease (RHD) within 24-62 h of infection. The rabbits were used to evaluate blood cell numbers and serum biochemistry every 6h, starting 12h after the inoculation of the caliciviruses. No significant changes in blood parameters were observed in most of the rabbits up to 18 h of infection. Severe leukopenia was seen 6h before death of the infected rabbits; both heterophils and lymphocytes contributed to the decrease in circulating white blood cells. Platelets were also severely decreased in number. Marked enhancement in liver enzymes was seen 6-12 h before death of the infected rabbits. There was also evidence both for cholestasis, as expressed by the elevated levels of direct (conjugated) bilirubin, and for hypoglycemia, an alteration that it is likely to contribute for the seizures that rabbits show during the late stages of RHD. Liver ultrastructure of rabbits that died from RHD revealed extensive hepatocyte vacuolization, severe changes in mitochondrial structure, and depletion of glycogen granules. We conclude that: (i) severe leukopenia characterizes the final hours of calicivirus-induced RHD; (ii) hypoglycemia and cholestasis precede death of rabbits from RHD; (iii) the kinetics of liver enzymes allows an accurate prediction of the time of death of rabbits from calicivirus-induced RHD.

Liver enzymes and ultrastructure in rabbit haemorrhagic disease (RHD).

Rabbit haemorrhagic disease (RHD) is caused by a calicivirus infection that kills most adult rabbits 24-72 h after viral inoculation. Two liver enzymes (AST, aspartate aminotransferase, and ALT, alanine aminotransferase) were monitored in blood samples of calicivirus-infected rabbits during the short course of RHD. Values of AST were used to differentiate three stages of hepatocellular degeneration in RHD: mild (up to 20-fold increase in AST), moderate (150-200-fold elevation of AST) and severe (more than 1000-fold elevation in AST). Liver samples of rabbits from these three biochemical stages of hepatocellular degeneration of RHD were studied by transmission electron microscopy to define the fine structure of the hepatocytes. In the mild hepatocellular degeneration there was proliferation (microvesiculation) of the smooth endoplasmic reticulum and swelling of mitochondria into spheroid bodies with loss of cristae. In moderate hepatocellular degeneration, vacuolization of cytoplasm and mitochondrial damage continued to be present, and there was also formation of autophagic vesicles. In the severe hepatocellular degeneration of RHD, the altered mitochondria also showed loss of density of their matrix; rupture of cytoplasmic vacuoles led to the formation of large vesicles. Marked depletion of liver glycogen was also found in this late stage of RHD. These data offer a correlation between biochemical and cytological features of the liver during the hepatocellular degeneration of RHD.

Leukocyte-hepatocyte interaction in calicivirus infection: differences between rabbits that are resistant or susceptible to rabbit haemorrhagic disease (RHD).

Calicivirus infection is lethal for adult rabbits, whereas young rabbits (less than 8-weeks-old) are resistant to the same infectious agent. The virus replicates in the liver and causes a fulminant hepatitis in adult rabbits leading to rabbit haemorrhagic disease (RHD); this is in contrast with the mild and transient hepatitis observed in infected young rabbits. We have used electron microscopy to compare liver leukocyte infiltrates between young (resistant) and adult (susceptible) rabbits, 36-48 h after inoculation of the animals with caliciviruses. In adult rabbits, liver infiltrates were made up mostly of heterophils, and they were located near hepatocytes showing severe cellular damage. In contrast, liver leukocyte infiltrates of RHD-resistant young rabbits were dominated by lymphocytes that depicted membrane contacts with the cell surface of undamaged hepatocytes. We conclude that: (i) the cellular inflammatory response of the liver to calicivirus infection is different in rabbits that are susceptible (adult) or resistant (young) to RHD; (ii) leukocyte infiltration of the adult liver by heterophils is probably directed at the removal of dead hepatocytes, whereas the liver lymphocytic infiltration of young rabbits suggests the expression of viral antigens on the surface of liver cells of the RHD-resistant animals.

Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors.

BACKGROUND: Conventional experiments in small scale are often performed in a 'Black Box' fashion, analyzing only the product concentration in the final sample. Online monitoring of relevant process characteristics and parameters such as substrate limitation, product inhibition and oxygen supply is lacking. Therefore, fully equipped laboratory-scale stirred tank bioreactors are hitherto required for detailed studies of new microbial systems. However, they are too spacious, laborious and expensive to be operated in larger number in parallel. Thus, the aim of this study is to present a new experimental approach to obtain dense quantitative process information by parallel use of two small-scale culture systems with online monitoring capabilities: Respiration Activity MOnitoring System (RAMOS) and the BioLector device. RESULTS: The same 'mastermix' (medium plus microorganisms) was distributed to the different small-scale culture systems: 1) RAMOS device; 2) 48-well microtiter plate for BioLector device; and 3) separate shake flasks or microtiter plates for offline sampling. By adjusting the same maximum oxygen transfer capacity (OTRmax), the results from the RAMOS and BioLector online monitoring systems supplemented each other very well for all studied microbial systems (E. coli, G. oxydans, K. lactis) and culture conditions (oxygen limitation, diauxic growth, auto-induction, buffer effects). CONCLUSIONS: The parallel use of RAMOS and BioLector devices is a suitable and fast approach to gain comprehensive quantitative data about growth and production behavior of the evaluated microorganisms. These acquired data largely reduce the necessary number of experiments in laboratory-scale stirred tank bioreactors for basic process development. Thus, much more quantitative information is obtained in parallel in shorter time.

Transient decrease in blood heterophils and sustained liver damage caused by calicivirus infection of young rabbits that are naturally resistant to rabbit haemorrhagic disease.

Young rabbits are naturally resistant to rabbit haemorrhagic disease (RHD) caused by the same calicivirus that kills, within 3 days, nearly all adult animals. We have investigated changes in blood leukocytes, and in the morphology and biochemistry of the liver (the organ where caliciviruses replicate) of young rabbits undergoing benign infection by the RHD virus. Four-week-old rabbits were infected with a calicivirus inoculum having a titre of 2(12) haemagglutination units either sacrificed 18, 24, 48 and 72 h later, or kept for follow-up studies up to 21 days after inoculation. The infection caused an acute and transient decrease in blood heterophils, and sustained enhancement in hepatic transaminases. Inflammatory infiltrates of the liver were seen in all animals after 24 h of infection; they had a predominant midlobular location. Hepatocytes could present different degrees of cell damage, including cell death; these lesions were limited to the liver cells located around the inflammatory infiltrates. Liver transaminases peaked 24-48 h after calicivirus infection; this was the same timing when liver infiltration and hepatocyte damage were more evident. No alterations of other parameters of liver biochemistry were observed. We conclude that calicivirus infection of young rabbits causes a subclinical disorder characterised by an acute and transient decrease in circulating heterophils, and focal liver damage that is expressed by intralobular infiltration by heterophils, initially, and, later on, by mononuclear cells. Our finding of persistence of increased values of liver transaminases suggests chronicity of the infection in young rabbits. We propose that, although resistant to RHD, young rabbits infected by calicivirus may be long-term carriers of the infectious agent and, thus, become a major source of transmission of the virus.

Neutron radiography and other NDE tests of main rotor helicopter blades.

A few nondestructive examination (NDE) techniques are extensively being used worldwide to investigate aircraft structures for all types of defects. The detection of corrosion and delaminations, which are believed to be the major initiators of defects leading to aircraft structural failures, are addressed by various NDE techniques. In a combined investigation by means of visual inspection, X-ray radiography and shearography on helicopter main rotor blades, neutron radiography (NRad) at SAFARI-1 research reactor operated by Necsa, was performed to introduce this form of NDE testing to the South African aviation industry to be evaluated for applicability. The results of the shearography, visual inspection and NRad techniques are compared in this paper. The main features and advantages of neutron radiography, within the framework of these investigations, will be highlighted.

Early inflammatory response of young rabbits attending natural resistance to calicivirus (RHDV) infection.

Young rabbits (i.e. up to 4 weeks of age) are naturally resistant to infection by rabbit haemorrhagic disease virus (RHDV), the same calicivirus that kills more than 90% of adult rabbits in 3 days or less. To characterize this fascinating model of age-related natural resistance to viral infection, we have studied the kinetics (from 6h up to 7 days) of cytokines and of leukocyte subpopulations in the liver (the target organ for calicivirus replication) and spleen (host systemic response) of RHDV infected young rabbits. Infection was associated with early (6h) elevation of proinflammatory cytokines (TNF-α, IL-1, IFN-α, IFN-γ, IL-6, IL-8). We found that all three major leukocyte subpopulations (macrophages, B and T lymphocytes) were increased in the liver 48h after the RHDV inoculation. At 7 days of infection, B and T lymphocytes were still elevated in the liver of the rabbits. In the spleen, both macrophages and B lymphocytes (but not T cells) were also enhanced. At 7 days, anti-RHDV specific antibodies were present in sera of all young rabbits infected by the virus. We conclude that natural resistance of young rabbits to RHDV infection is associated with a rapid and effective inflammatory response by the liver, with few hepatocytes being infected, and also with a sustained elevation in local and systemic B and T cells.

Effect of experimental chronic renal failure upon the production of urea, as measured by the liver arginase activity in rats.

A significant diminution in liver arginase activity of chronic renal failure uremic rats is described, thus implying that the experimental finding of an increased urea production is not due to a depressed arginase activity.

Effect of experimental chronic renal failure upon the synthesis of thyroid hormones in rats.

The authors utilized two groups of adult male rats to study the effect of chronic-uremic toxemia on the synthesis of thyroid hormones. The control group was sham operated, while the other group had about 70--80% of the left kidney tissue surgically excised and a total right nephrectomy performed 10 days later. At the 90th day after the second operation, 8 muCi of 131I were injected intraperitoneally and 24 h later the thyroid was excised and hydrolyzed enzymatically for a period of 14 h at 37 degrees C. The percentages of iodinated components were then calculated after radiochromatographic separation on paper. The group of experimental rats by this time showed significant increases of plasma urea and creatinine, and an absence of acidosis. The results suggest that monoidotyrosine concentration is increased and diiodothyronine decreased in the experimental group of rats with renal failure. This might indicate a toxic metabolic blockage in the transformation of monoiodotyrosine to diiodothyronine.

Glucagon stimulation test for the diagnosis of GH deficiency in adults.

The insulin tolerance test (ITT) is considered the test of choice for the diagnosis of GH deficiency (GHD). However, in patients with contraindications to ITT, alternative provocative tests must be used with appropriate cut-offs. The glucagon stimulation test has proved to be a safe, low-cost and effective means of stimulating GH secretion, and therefore can be considered as a suitable alternative to the ITT. We have studied the GH response to the glucagon test in 33 patients with known pituitary disease, 12 males and 21 females, aged between 21 and 60 yr (41.18 +/- 9.47 yr); 5 had isolated GHD and 28 had panhypopituitarism. We also evaluated a control group of 25 individuals, matched for age and sex (8 males and 17 females), aged between 20 and 60 yr (39.28 +/- 12.10 yr). They were selected via the ITT if their peak GH response was > 5.0 ng/ml. GH peak after glucagon was significantly lower in the group of patients compared to the control group (0.49 +/- 0.85 vs 8.69 +/- 5.85 ng/ml, p = 0.0001). Receiver-operating characteristic (ROC) plot analyses of the control and GHD group showed an area under the curve of 0.982 for GH peak response to glucagon. The response value of 3.0 ng/ml showed the best pair of sensitivity (97%)/specificity (88%), and was chosen as the cut-off defining GHD. After evaluation of positive predictive values (PPV) and negative predictive values (NPV) through simulation of different prevalences of the disease, we concluded that the cut-off point of 3.00 ng/ml maximizes both PPV and NPV (100%). In conclusion, we have shown that the glucagon stimulation test has a good performance and great diagnostic accuracy for the diagnosis of GHD.

Effect of experimentally induced chronic renal failure upon the behavior of rats.

Two groups of rats were submitted to a 2-hour training session under a free operant avoidance of shock contingency 23 weeks after surgery, which provoked a state of chronic renal failure (experimental group) or after a sham operation (control group). Subjects in the control group had superior performances than those from the experimental group when total of shocks received and total numbers of avoidance and escape responses were compared. Results of biochemical blood analyses obtained from samples taken immediately after the avoidance training session indicate that the experimental group showed higher plasma urea concentration, higher levels of creatinine and osmolarity. The data show that the procedure used to provoke chronic renal failure was successful, and results in general confirm and extend findings relative to the effects of acute renal failure on behavior.

Variations in the anatomical organization of the human aortic arch. A study in a Portuguese population.

We have studied the anatomical features of human aortic arches in a sample of the Portuguese population using postmortem measurements of the arches and their branches in 33 cadavers. We found that there is a significant minority of individuals (18% of the total) who did not present the usual 3-branched aortic arch; interestingly, all 2-branched arches were seen in women and all 4-branched arches were observed in men. The arterial branches could spring either from the ascending segment of the arch (61% of total) or from its horizontal segment (39% of total). Our measurements showed that the springing of the branches was associated with the narrowing of the aortic arch, so that the difference in the diameter of the aortic arch between the origin of the first and last branches could reach up to 11 millimeters. We found that the relative caliber of the arterial branches of the aortic arch depended on the position of its origin at the arch: in aortic arches with branches that were originated at the horizontal segment of the arch the first arterial branch was always the widest of the three, whereas in aortic arches with branches coming out at the ascending segment of the arch the widest arterial branch was the third one. Our data support the view that there is a wide spectrum of variations in the anatomical arrangement of the aortic arch in humans, and offer information on the prevalence of aortic arch variations in a Portuguese population.

A new plethysmograph for detecting arterial pulse waves in rat tail.

A new plethysmograph for detecting arterial pulse waves in the rat tail is described. With the objective of indirectly measuring the arterial blood pressure in small animal tails an easy-to-build plethysmographic chamber is presented. Essentially in this apparatus the atmospheric air insulated in the chamber is used as the transmitter of the volume changes caused by the arterial flow. By this methodology details of the pulse wave morphology are clearly visible, including the dicrotic notch. It is demonstrated that his chamber combines simplicity with accuracy.