Pre-grafting histological studies of skin grafts cryopreserved in α helix antarctic yeast oriented antifreeze peptide (Afp1m).

A number of living creatures in the Antarctic region have developed characteristic adaptation of cold weather by producing antifreeze proteins (AFP). Antifreeze peptide (Afp1m) fragment have been designed in the sequence of strings from native proteins. The objectives of this study were to assess the properties of Afp1m to cryopreserve skin graft at the temperature of -10 °C and -20 °C and to assess sub-zero injuries in Afp1m cryopreserved skin graft using light microscopic techniques. In the present study, a process was developed to cryopreserve Sprague-Dawley (SD) rat skin grafts with antifreeze peptide, Afp1m, α-helix peptide fragment derived from Glaciozyma antractica yeast. Its viability assessed by different microscopic techniques. This study also described the damages caused by subzero temperatures (-10 and -20 °C) on tissue cryopreserved in different concentrations of Afp1m (0.5, 1, 2, 5 and 10 mg/mL) for 72 h. Histological scores of epidermis, dermis and hypodermis of cryopreserved skin grafts showed highly significant difference (p < 0.01) among the different concentrations at -10 and -20 °C. In conclusion, the integrity of cryopreserved skin grafts with lower concentrations of Afp1m (0.5, 1 and 2 mg/mL) or at -20 °C was not maintained. The present study attested that Afp1m is a good cryoprotective agent for the cryopreservation of skin graft. Higher Afp1m concentrations (5 and 10 mg/mL) at -10 °C found to be suitable for the future in vivo study using (SD) rat skin grafts.

Molecular detection of Haemophilus parasuis serotypes 4, 5 or 12 and 13 in Peninsular Malaysia.

Although the economic importance of Haemophilus parasuis infection causing Glasser's disease is prevalent throughout pig farms in Peninsular Malaysia, there is a dearth of knowledge on its actual nature. In this study, a multiplex PCR was performed to screen for three major predominant virulent strains of H. parasuis, which are serotypes 4, 5 or 12 and 13. A total of 175 tissues or bodily fluid samples of various parts were collected from diseased animals from October, 2016 to February, 2018; with total of 62.9% positive detection of H. parasuis. The highest detection was found to be in the pericardial sac fibrin (90.9%) followed by pleural fibrin, lung, pleural fluid, tonsil, pericardial sac, peritoneal fluid, abdominal fibrin, joint fluid, brain and pericardium. Serotype 13 was the highest (40/110) followed by serotype 4(37/110), serotype 5(31/110) and 12 samples were nontypable (12/110). The presence of untypable serotype also drives to further identification of other serotypes in Malaysia.

Toxoplasma gondii infection in native village chickens (Gallus domesticus) in Selangor and Melaka, Malaysia.

Toxoplasmosis is a worldwide zoonosis caused by the protozoa Toxoplasma gondii which affects human and animals. Village chickens (Gallus domesticus) most commonly known as Ayam Kampung or free-range chickens, have been suggested to play a role in the epidemiology of toxoplasmosis. This study determines the presence of T. gondii in the village chicken populations in two states of Malaysia. A total of 50 serum samples from the chickens from Selangor (n=20) and Melaka (n=30) were collected and analysed using commercial serological kits. T. gondii antigen was detected in 20% (Selangor 30%; Melaka 13%) samples using ELISA test and anti-T. gondii antibody was detected in all positive ELISA samples using the indirect haemagglutination test (IHAT). Histopathological examination revealed tissue changes such as inflammation and degeneration in brain and liver of seropositive chickens. This is the first report of T. gondii infection in the village chickens in Malaysia.

Molecular detection of Haemophilus parasuis serotypes 4, 5 or 12 and 13 in Peninsular Malaysia

@#Although the economic importance of Haemophilus parasuis infection causing Glasser’s disease is prevalent throughout pig farms in Peninsular Malaysia, there is a dearth of knowledge on its actual nature. In this study, a multiplex PCR was performed to screen for three major predominant virulent strains of H. parasuis, which are serotypes 4, 5 or 12 and 13. A total of 175 tissues or bodily fluid samples of various parts were collected from diseased animals from October, 2016 to February, 2018; with total of 62.9% positive detection of H. parasuis. The highest detection was found to be in the pericardial sac fibrin (90.9%) followed by pleural fibrin, lung, pleural fluid, tonsil, pericardial sac, peritoneal fluid, abdominal fibrin, joint fluid, brain and pericardium. Serotype 13 was the highest (40/110) followed by serotype 4(37/110), serotype 5(31/110) and 12 samples were nontypable (12/110). The presence of untypable serotype also drives to further identification of other serotypes in Malaysia.

Toxoplasma gondii infection in native village chickens (Gallus domesticus) in Selangor and Melaka, Malaysia

@#Toxoplasmosis is a worldwide zoonosis caused by the protozoa Toxoplasma gondii which affects human and animals. Village chickens (Gallus domesticus) most commonly known as Ayam Kampung or free-range chickens, have been suggested to play a role in the epidemiology of toxoplasmosis. This study determines the presence of T. gondii in the village chicken populations in two states of Malaysia. A total of 50 serum samples from the chickens from Selangor (n=20) and Melaka (n=30) were collected and analysed using commercial serological kits. T. gondii antigen was detected in 20% (Selangor 30%; Melaka 13%) samples using ELISA test and anti-T. gondii antibody was detected in all positive ELISA samples using the indirect haemagglutination test (IHAT). Histopathological examination revealed tissue changes such as inflammation and degeneration in brain and liver of seropositive chickens. This is the first report of T. gondii infection in the village chickens in Malaysia.

Histological and mechanical evaluation of antifreeze peptide (Afp1m) cryopreserved skin grafts post transplantation in a rat model.

The objective of this study was to evaluate the use of Afp1m as a cryopreservative agent for skin by examining the transplanted skin histological architecture and mechanical properties following subzero cryopreservation. Thirty four (34) rats with an average weight of 208 ±â€¯31 g (mean ±â€¯SD), were used. Twenty four (n = 24) rats were equally divided into four groups: (i) immediate non-cryopreserved skin autografts (onto same site), (ii) immediate non-cryopreserved skin autografts (onto different sites), (iii) skin autografts cryopreserved with glycerol for 72 h and (iv) skin autografts cryopreserved with Afp1m for 72 h at -4 °C. Rounded shaped full-thickness 1.5-2.5 cm in diameter skin was excised from backs of rats for the autograft transplantation. Non-cryopreserved or cryopreserved auto skin graft were positioned onto the wound defects and stitched. Non-transplanted cryopreserved and non-cryopreserved skin strips from other ten rats (n = 10) were allowed for comparative biomechanical test. All skin grafts were subjected to histological and mechanical examinations at the end of day 21. Histological results revealed that tissue architecture especially the epidermal integrity and dermal-epidermal junction of the Afp1m cryopreserved skin grafts exhibited better histological appearance, good preservation of tissue architecture and structural integrity than glycerolized skin. However, there was no significant difference among these groups in other histological criteria. There were no significant differences among the 4 groups in skin graft mechanical properties namely maximum load. In conclusion, Afp1m were found to be able to preserve the microstructure as well as the viability and function of the skin destined for skin transplantation when was kept at -4 °C for 72 h.

The induction of aryl hydrocarbon receptor (AHR) in immune organs of developing chicks by polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons are pollutants which are persistent in nature. The aryl hydrocarbon receptor is a ligand-activated cytosolic transcription factor activated by xenobiotics. The objective was to isolate and identify AHR mRNA transcript in immune organs of developing chicks and to interpret the correlation between AHR induction and dose of PAHs. Specific pathogen free embryonated eggs on day nine were inoculated with solutions of pyrene, phenanthrene, and fluoranthene dissolved in tricaprylin (vehicle) through the allantoic route at three dose levels: 0.2 mg/kg, 2 mg/kg, and 20 mg/kg. A 650 base pair product was observed by RNA extraction and reverse transcription PCR from thymus, bursa of Fabricius and spleen on 21st day. When AHR concentration was analyzed by ELISA in these organs, pyrene showed maximum potency in inducing AHR in thymus. Fluoranthene made highest concentration of AHR in bursa of Fabricius. None of these chemicals caused an increase in AHR concentration in spleen.

DNA damage and adduct formation in immune organs of developing chicks by polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are persistent pollutants and chemically a class of structurally similar chemical compounds characterized by the presence of fused aromatic rings. This research was undertaken to find out immunotoxic effects produced by pyrene, phenanthrene and fluoranthene. These chemicals were injected into developing chicks at three dose levels (0.2, 2 and 20 mg per kg) through allantioc route to rule out possible mechanisms involved in immunotoxicity. DNA adduct produced by PAHs in immune organs were analyzed by DNA adduct enzyme-linked immunosorbent assay (ELISA) kit and DNA damage was assessed by comet assay. A significant increase in the DNA adduct levels was found in thymus and bursa in 2 mg and 20 mg dose levels of pyrene, fluoranthene and phenanthrene treated groups, whereas those in spleen simulated the value of controls. Comet assay indicated that PAHs especially pyrene, fluoranthene and phenanthrene were capable of inducing increased level of comet parameters in thymus at all the dose levels. Bursa of Fabricius and spleen also showed a gradual rise in comet parameters corresponding to all dose levels, but the increase was more marked as in thymus. Thus, it can be concluded that DNA adducts produced by PAHs lead to single-strand breaks and reduced DNA repair, which ultimately begin a carcinogenic process. Hence, this experiment can be considered as a strong evidence of genotoxic potential of PAHs like pyrene, phenanthrene and fluoranthene in developing chicks.

The use of rats and mice as animal models in ex vivo bone growth and development studies.

In vivo animal experimentation has been one of the cornerstones of biological and biomedical research, particularly in the field of clinical medicine and pharmaceuticals. The conventional in vivo model system is invariably associated with high production costs and strict ethical considerations. These limitations led to the evolution of an ex vivo model system which partially or completely surmounted some of the constraints faced in an in vivo model system. The ex vivo rodent bone culture system has been used to elucidate the understanding of skeletal physiology and pathophysiology for more than 90 years. This review attempts to provide a brief summary of the historical evolution of the rodent bone culture system with emphasis on the strengths and limitations of the model. It encompasses the frequency of use of rats and mice for ex vivo bone studies, nutritional requirements in ex vivo bone growth and emerging developments and technologies. This compilation of information could assist researchers in the field of regenerative medicine and bone tissue engineering towards a better understanding of skeletal growth and development for application in general clinical medicine.Cite this article: A. A. Abubakar, M. M. Noordin, T. I. Azmi, U. Kaka, M. Y. Loqman. The use of rats and mice as animal models in ex vivo bone growth and development studies. Bone Joint Res 2016;5:610-618. DOI: 10.1302/2046-3758.512.BJR-2016-0102.R2.

Immunohistochemical study of endocrine cells in the gastrointestinal tract of the barking deer, Muntiacus muntjak.

The morphology, distribution and relative frequencies of endocrine cells in the gastrointestinal tract of the barking deer (Muntiacus muntjak) were investigated. The immunohistochemical method employed used seven types of antisera against chromogranin, serotonin, gastrin, cholecystokinin, somatostatin, glucagon and insulin. Chromogranin and serotonin immunoreactive (IR) cells were found throughout the gastrointestinal tract. Conversely, gastrin-IR cells were only found in the oxyntic gland, pyloric gland and duodenum, while cholecystokinin-IR and somatostatin-IR cells were detected in the oxyntic gland, pyloric gland and small intestines. Somatostatin-IR cells were also seen in the caecum. Glucagon-IR cells were found in all parts of the gastrointestinal tract apart from the colon and rectum. No insulin-IR cells were found in the gastrointestinal tract of this species. The cells in the small intestine were generally spindle shaped with long cytoplasmic processes ending in the lumen (open type), while in the stomach and large intestine, they were occasionally round or spherical in shape (closed type). An uncommon distribution pattern of endocrine cells in the gastrointestinal tract of the barking deer was noted for cholecystokinin- and glucagon-IR cells.

Efficacy of Nigella sativa in alleviating benzo[a]pyrene-induced immunotoxicity in broilers.

The immune response of broiler chickens exposed to intra-tracheal (i.t.) administration of benzo[a]pyrene (BaP) with and without Nigella sativa (Ns) supplementation was investigated. A total of 120 day-old chicks were divided into four groups comprising 30 birds each, into a control, Ns, BaP, and BaP+Ns group. Immune responses to Newcastle disease (ND) were evaluated by haemagglutination inhibition (HI), phytohaemagglutinin (PHA) skin test and carbon clearance assay (CCA). In most instances, there was a significant increase (p<0.05) in the ND-HI antibody titers, PHA skin-swelling response and phagocytic activity in the BaP + Ns group compared to that of the BaP group. Likewise, organ weight and indices of the spleen, bursa of Fabricius and thymus of birds from the BaP + Ns group were also higher (p<0.05) than that of the BaP group from day 1 until day 21. It is concluded that exposure to BaP may exert adverse effects on the immune system of broilers which may increase their susceptibility to disease, and Ns supplementation significantly reduces these alterations.

Histomorphology of the stomach, proventriculus and ventriculus of the red jungle fowl.

The cranial chamber (proventriculus) and caudal chamber (ventriculus) of the stomach of the Red jungle fowl (Gallus gallus spadiceus) were examined by means of light microscopy. Both chambers presented folds of the tunica mucosa lined by a simple prismatic epithelium that was positive for neutral mucin. Simple tubular glands occupied the lamina propria of both chambers; in the ventriculus of older birds, they showed a coiled base. These ventricular glands were lined by simple cuboidal cells represented by the chief cells and a few large basal cells. The luminal and tubular koilin rodlets and folds of the ventriculus were positive to periodic acid Schiff (PAS) stain. The proventricular glands were situated between the inner and outer layers of the lamina muscularis mucosae. Cells lining the tubulo-alveolar units of the proventricular glands showed a dentate appearance. Vacuoles were not observed, and the cells were negative for Alcian-PAS stain. The tunica submucosa was very thin in the proventricular wall. In the ventriculus, it was not separated from the lamina propria owing to the absence of any lamina muscularis mucosae. The tunica muscularis of the proventriculus was formed by a thick inner layer of circular smooth muscle fibres and a thin outer layer of longitudinal fibres. In addition to these layers, oblique muscle fibres formed the most internal layer of the tunica muscularis in the ventriculus.

Effects of Momordica charantia on pancreatic histopathological changes associated with streptozotocin-induced diabetes in neonatal rats.

The aim of this research was to determine the effects of Momordica charantia (MC) fruit aqueous extract on pancreatic histopathological changes in neonatal STZ-induced type-II diabetic rats. Diabetes mellitus was induced in one day Sprague-Dawley neonatal rats using a single intrapretoneal injection of streptozotocin (STZ) (85 mg/kg body weight) and monitored for 12 weeks thereafter. The diabetic rats were separated into three groups, as follows: the diabetic control group (i.e. nSTZ), the diabetic group (i.e. nSTZ/M) - which was orally given 20 mg/kg of MC fruit extract, and the diabetic group (i.e. nSTZ/G) - that was treated with glibenclamide, 0.1 mg/kg for a period of four weeks. At the end of treatment, the animals were sacrificed and blood samples were collected from the saphenous vein to measure the blood glucose and serum insulin level. The pancreatic specimens were removed and processed for light microscopy, electron microscopy examination and immunohistochemical study. The results of this study showed that MC fruit aqueous extract reduced the blood glucose level as well as glibenclamide and increased the serum insulin level in the treated diabetic rats (P<0.05). The fruit extract of MC alleviated pancreatic damage and increased the number of ß-cells in the diabetic treated rats (P<0.05). Our results suggest that oral feeding of MC fruit extract may have a significant role in the renewal of pancreatic ß-cells in the nSTZ rats.

Morphological study of the lingual papillae in the barking deer, Muntiacus muntjak.

The morphology of the tongue of the adult barking deer, Muntiacus muntjak, was examined by light and scanning electron microscopy. The result showed that the tongue of the barking deer was elongated with a rounded apex. Four types of lingual papillae were observed: filiform, fungiform, vallate and large conical papillae. The filiform papillae represented the most numerous types of lingual papillae. The fungiform papillae were distributed among the filiform papillae on the rostral and the body portions of the tongue. Ten to thirteen vallate papillae were distributed on both sides of the lingual prominence among the large conical papillae. Histologically, both the fungiform and vallate papillae contain taste buds in the epithelial layer. The distribution and types of lingual papillae found in the barking deer are similar to those in the other species that belong to the family Cervidae.

Histological study of the parotid and mandibular glands of barking deer (Muntiacus muntjak) with special reference to the distribution of carbohydrate content.

We investigated the histology and carbohydrate content of the parotid and mandibular glands of the barking deer (Muntiacus muntjak). Three adult males were used. Paraffin wax sections of the glands were stained with haematoxylin and eosin (HE), alcian blue (AB), pH 2.5 and periodic acid Schiff (PAS). The acinar cells of the parotid gland were serous, whereas those of the mandibular gland were of the mixed type. The acini of the mandibular gland comprised serous and mucous cells with the mucous type predominating. AB and PAS staining showed high concentrations of acidic and neutral carbohydrates in the mucous cells, but not in the serous cells of the mandibular gland. These carbohydrates were also found in moderate-to-high concentrations in the secreted material in the mandibular duct lumen. However, these carbohydrates were not found in acinar cells of the parotid gland or in the serous cells of the mandibular gland. Thus, carbohydrates in the saliva of the barking deer appear to be produced mainly by the mucous cells of the mandibular glands.

Pulmonary modulation of benzo[a]pyrene-induced hemato- and hepatotoxicity in broilers.

Aftermath in several air pollution episodes with high concentrations of polycyclic aromatic hydrocarbons did not significantly affect health and performance of broilers despite its renowned sensitivity to polycyclic aromatic hydrocarbons. The aim of the study was to elucidate the previous lack of response in birds exposed to such severe episodes of air pollution. Benzo[a]pyrene (BaP) was used to simulate the influence of air pollution on hematology, selected organ function, and oxidative stress in broilers. One-day-old chicks were assigned to 5 equal groups composed of a control group, tricaprylin group, and 3 groups treated with BaP (at 1.5 microg, 150 microg, or 15 mg/kg of BW). The BaP was intratracheally administered to 1-d-old chicks for 5 consecutive days. The hematology, liver and kidney function, P450 activity, and malondialdehyde level especially in the group receiving 15 mg of BaP/kg of BW demonstrated evidence of hemato- and hepatoxicity via BaP-induced oxidative stress. The deleterious effect of exposure to high concentration of BaP in broiler chickens was probably due to the anatomy of this species and the half-life of BaP. Although the effect of BaP may be transient or irreversible, pathogen challenges faced during the period of suppression may prove fatal.

Tissue engineering approach to repair abdominal wall defects using cell-seeded bovine tunica vaginalis in a rabbit model.

The aim of this study was to engineer skeletal muscle tissue for repair abdominal wall defects. Myoblast were seeded onto the scaffolds and cultivated in vitro for 5 days. Full thickness abdominal wall defects (3 x 4 cm) were created in 18 male New Zealand white rabbits and randomly divided into two equal groups. The defects of the first group were repaired with myoblast-seeded-bovine tunica vaginalis whereas the second group repaired with non-seeded-bovine tunica vaginalis and function as a control. Three animals were sacrificed at 7th, 14th, and 30th days of post-implantation from each group and the explanted specimens were subjected to macroscopic and microscopic analysis. In every case, seeded scaffolds have better deposition of newly formed collagen with neo-vascularisation than control group. Interestingly, multinucleated myotubes and myofibers were only detected in cell-seeded group. This study demonstrated that myoblast-seeded-bovine tunica vaginalis can be used as an effective scaffold to repair severe and large abdominal wall defects with regeneration of skeletal muscle tissue.

Mineral and physichocemical characterization of cockle (Anadara granosa) shells as an alternative biomaterial for bone tissue engineering.

This study was conducted based on the hypothesis that mineral and physicochemical properties of cockle shells similarly resemble the properties of corals (Porites sp.). Hence, the mineral and physicochemical evaluations of cockle shells were conducted to support the aforementioned hypothesis. The results indicated that cockle shells and coral exoskeleton shared similar mineral and physicochemical properties.

Effect of preservation methods on the performance of bovine pericardium graft in a rat model.

This study investigates the effect of preservation methods on the performance of bovine parietal pericardium grafts in a rat model. Mid-ventral full thickness abdominal wall defects of 3 x 2.5 cm in size were created in 90 male Sprague-Dawley rats (300-400 g), which were divided into three groups of 30 rats each. The abdominal defects of group one and two were repaired with lyophilized and glycerolized bovine pericardium grafts, while the defects of group three were repaired with expanded polytetrafluoroethylene (ePTFE) Mycro Mesh as a positive control. Another group of 30 rats underwent sham operation and was used for comparison as negative control. Each group of rats (n = 30) was divided into five subgroups (n = 6) and killed at 1, 3, 6, 9 and 18 weeks post-surgery for gross and morphological evaluations. The rats tolerated the surgical procedure well with a total mortality of 0.05%. No serious post-operative clinical complications or signs of rejection were encountered. Adhesions between the grafts and the underlying visceral organs observed in the study were mostly results of post-surgical complications. Glycerol preservation delayed degradation and replacement of the grafts, whereas lyophilization caused early resorption and replacement of the grafts. The glycerolized grafts were replaced with thick dense fibrous tissue, and the lyophilized grafts were replaced with thin loose fibrous tissue. The healing characteristic of the bovine pericardium grafts was similar to those of the sham-operated group, and quite different from those of the ePTFE Mycro Mesh. The outcome of the present study confirmed the superiority of glycerolized bovine pericardium grafts over its lyophilized counter part.

Effect of freeze-drying and gamma irradiation on biomechanical properties of bovine pericardium.

Freeze-drying and gamma irradiation are the techniques widely use in tissue banking for preservation and sterilization of tissue grafts respectively. However, the effect of these techniques on biomechanical properties of bovine pericardium is poorly known. A total of 300 strips of bovine pericardium each measured 4 cm x 1 cm were used in this study to evaluate the effect of freeze-drying on biomechanical properties of fresh bovine pericardium and the effect of gamma irradiation on biomechanical properties of freeze-dried bovine pericardium. The strips were divided into three equal groups, which consist of 100 strips each group. The three groups were fresh bovine pericardium, freeze-dried bovine pericardium and irradiated freeze-dried bovine pericardium. The biomechanical properties of the pericardial strips were measured by a computer controlled instron tensiometer while the strips thickness was measured by Mitutoyo thickness gauge. The results of the study revealed that freeze-drying has no significant (p > 0.05) effect on the tensile strength, Young's modulus (stiffness) and elongation rate of fresh bovine pericardium. Irradiation with 25 kGy gamma rays caused significant decreased in the tensile strength, Young's modulus and elongation rate of the freeze-dried pericardium. However, gamma irradiation has no significant effect on the thickness of freeze-dried bovine pericardium, while freeze-drying caused significant decreased in the thickness of the fresh bovine pericardium. The outcome of this study demonstrated that freeze-drying has no significant effect on the biomechanical properties of fresh bovine pericardium, and gamma irradiation caused significant effect on the biomechanical properties of freeze-dried bovine pericardium.