Effect of Diazepam on Proliferation of Chondrocytes in Intermediate and Deep Zones of Patellar Articular Cartilage in Adult Male Mice.

OBJECTIVE: To determine the effect of diazepam on proliferation of chondrocytes in intermediate and deep zones of degenerating patellar articular cartilage in Balb/c mice. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Department of Anatomy, College of Physicians and Surgeons Pakistan, Regional Centre, Islamabad, Pakistan, from June 2015 to May 2016. METHODOLOGY: There were two groups of adult male mice as control group A and experimental group B. Group A having 90 mice was further divided into three subgroups having 30 mice in each subgroups. In subgroup A1 having 30 mice were treated normally. Subgroup A2 mice were kept immobilised for 20 days. Mice in subgroup A3 were given normal saline intraperitoneally during next 30 days of remobilisation and sacrificed. Among the mice in group B, normal saline was replaced with diazepam at the dose of 4 mg/kg for 30 days. Control subgroups and experimental group were compared. Data was analysed quantitatively using SPSS Version 16. RESULTS: Significant decrease in mean thickness of combined zone (66.69 ± 2.77µm) and chondrocytes count (5.61±0.76) were seen in subgroup A2 mice than subgroup A1 (77.54 ± 3.4µm) and (5.61 ± 0.76), respectively (p<0.001). Subgroup A3 showed significantly increased mean thickness (95.45 ± 5.17µm) and chondrocytes count (7.31 ± 0.44) than subgroup A2 (P<0.001). No significant increase in mean thickness (96.46 ± 4.94µm) and chondrocytes count (7.35 ± 0.41) of group B were seen than subgroup A3 (p=0.01). CONCLUSION: Diazepam administration does not stimulate proliferation of chondrocytes of combined intermediate and deep zones of degenerating articular cartilage by acting on GABA A receptors.

Quantitative Analysis of the Area of the Apical Ectodermal Ridge in Chick Appendages Using Image-J.

OBJECTIVE: To determine the effect of sodium phenytoin on the apical ectodermal ridges (AER) of chick wing buds by using the software program Image-J. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Department of Anatomy, Regional Center, College of Physicians and Surgeons Pakistan (CPSP), Islamabad, from January 2014 to January 2015. METHODOLOGY: Sixty fertilised chicken eggs of 'Egyptian fayoumi' breed were selected and separated into experimental (B) and control (A) groups, each having 30 eggs. A single dose of 3.5 mg sodium phenytoin was injected into each egg of the experimental group. The controls were injected with the same volume of normal saline. Developing embryos were extracted 96 hours (day 4) after incubation and histological sections were cut at 5 µm thickness. These sections were stained with Feulgen Nuclear and Light Green. The area of apical ectodermal ridges of chick wing buds was calculated by employing Image-J and subjected to statistical analysis. RESULTS: The difference between the mean values of the area of apical ectodermal ridges of experimental and control groups, as calculated by Image-J, was found to be statistically insignificant. CONCLUSION: Change in the area of the apical ectodermal ridges in experimental chicks, following phenytoin exposure, was insignificant as proven on the basis of quantification by Image-J.

Quantitative volumetric analysis of a retinoic acid induced hypoplastic model of chick thymus, using Image-J.

OBJECTIVE: To assess the total volume change in a retinoic acid-induced, hypoplastic model of a chick thymus using Image-J. METHODS: This experimental study was carried out at the anatomy department of College of Physicians and Surgeons, Islamabad, Pakistan, from February 2009 to February 2010, and comprised fertilised chicken eggs. The eggs were divided into experimental group A and control group C. Group A was injected with 0.3µg of retinoic acid via yolk sac to induce a defective model of a thymus with hypoplasia. The chicks were sacrificed on embryonic day 15 and at hatching. The thymus of each animal was processed, serially sectioned and stained. The total area of each section of thymus was calculated using Image-J. This total area was summed and multiplied with the thickness of each section to obtain volume. RESULTS: Of the 120 eggs, there were 60(50%) in each group. Image analysis revealed a highly significant decrease in the volume of the chick thymus in the experimental group A than its matched control at the time of hatching (p=0.001). Moreover, volumetric depletion progressed with time, being substantially pronounced at hatching compared to the embryonic stage. CONCLUSIONS: The volume changes were significant and were effectively quantified using Image-J.

Effect of administration of glucose on body length and body weight of the chick embryos.

Objective: To assess the effect of glucose on body length and body weight of chick embryos. Methods: This experimental study was carried out at the College of Physicians and Surgeons Pakistan, Islamabad, from January 2013 to January 2014, and comprised chicken eggs. Fertilised eggs of Egyptian Fayyumi breed were injected with glucose (5% weight/volume solution) into egg albumen. The eggs were put in the incubator under standard conditions of temperature and humidity. Eggs were divided in two groups; control group A and experimental group B. Each group was subdivided in three subgroups. Eggs were opened on day 12 (A1, B1), day 15(A2, B2), and day 18(A3, B3) of incubation and the dissected-out embryos were compared with age-matched control subgroups. Effects of glucose were assessed by measuring the body weight and body length of embryos. Results: Of the 180 eggs, there were 30(16.67%) in each of the 6 subgroups. The mean body length was 6.527±0.086cm in A1 and 5.287±0.035 in B1 (p=0.001); 9.560±0.095 in A2 and 9.237±0.114 in B2 (p=0.033); and 13.919±0.093 in A3 and 16.117±0.103 in B3 (p=0.000). Similarly, the mean body weight was 4.374±0.071 in A1 and 3.676±0.007 in B1 (p=0.001); 10.814±0.214 in A2 and 11.009±0.339 in B2 (p=0.619); and 18.142±0.123 in A3 and 22.87±0.067 in B3 (p=0.000).. CONCLUSIONS: Administration of glucose resulted in initial growth retardation of developing embryos but later on there was significant growth acceleration as the age advanced.

The effect of ethanol vapour exposure on atrial and ventricular walls of chick embryos.

OBJECTIVE: To study the effects of ethanol vapour exposure on atrial and ventricular walls of heart in chick embryo. METHODS: The study design was experimental, conducted at Islamabad Centre of College of Physicians and Surgeons, Pakistan. One hundred and eighty chicken eggs were divided into two groups, experimental and control, of 90 eggs each. Each group was subdivided into three subgroups of 30 eggs each based on the day of sacrifice. Experimental group was exposed to ethanol vapours and then compared with age matched controls. RESULTS: The thickness of atrial and ventricular walls along with lengths of valvular cusps increased in hearts of day 7 and day 10 chick embryos in experimental group. There was thinning of walls and decreased length of valvular cusps in hearts of experimental chicks on hatching as compared to age matched controls. CONCLUSIONS: Ethanol vapour exposure during development causes cardiac and septal wall thickening during initial days of development followed by cardiac and septal wall thinning which is a classical picture of alcohol induced cardiomyopathies.

Does folinic acid ameliorates retinoic acid induced teratogenicity in chick embryo?

OBJECTIVE: To investigate the protective effect of folinic acid on the hatching ability and developmental defects in a retinoic acid-induced teratogenic model of chick embryo. METHODS: The experimental study was conducted at the Department of Anatomy, Regional Centre of the College of Physicians and Surgeons Pakistan, Islamabad, from February 2009 to February 2010. Chicken eggs were divided into two experimental groups and a control group. The first experimental group was injected with retinoic acid to induce a defective model, while the second experimental group was concomitantly injected folinic acid to observe its protective effects on retinoic acid-induced defects in the development and hatching process. Both groups were compared with the age-matched control group. RESULTS: A total of 90 fertilised eggs were divided into three groups. The experimental groups had significantly more delayed and assisted hatchings compared to the control group (p<0.05) but the difference between the experimental groups regarding the mode and day of hatching was insignificant (p>0.05). CONCLUSIONS: Irrespective of the presence of folinic acid, prenatal retinoic acid exposure significantly altered the hatchability characteristics in the experimental groups compared to the control.

Atrial and ventricular septal changes in ethanol vapour exposed chick embryos.

OBJECTIVE: To study the effects of ethanol vapour exposure on development of atrial and ventricular septa of chick embryo. METHODS: The experimental study was conducted at the College of Physicians and Surgeons, Islamabad, from 2006 to 2007. The experimental and control groups were further divided into three subgroups based on the day of sacrifice. The experimental group was exposed to ethanol vapours produced in a specially-designed vapour chamber and then compared with age-matched controls. RESULTS: There were 90 eggs in each of the two groups. The development of inter-ventricular septum completed at day 7 of development in chick embryo. Ethanol vapour exposure produced a small discontinuity at day 10 of development in a chick embryo which may be labelled as ventricular septal defect since ventricular development is completed by day 7. Interatrial septum formed till day 7 with small perforations which persisted till hatching. CONCLUSIONS: Ethanol vapour exposure may lead to ventricular septal defect.

Role of oxidative injury in affecting the foetal & placental weights on exposure to tobacco smoke.

OBJECTIVE: To investigate the role of oxidative injury in affecting foetal and placental weights in mice by exposing them to tobacco smoke with or without supplementation with antioxidants. METHODS: The randomized control trial of pregnant mice at day one of gestation was conducted at Anatomy Department CPSP Regional Center Islamabad, from March 2005 to October 2005. The mice were divided into three groups: Group C had controls, while the two other groups, groups S and SV were exposed to secondary tobacco smoke in a whole body exposure chamber with and without supplementation with vitamins respectively. At term, the animals were sacrificed and the placentae and foetuses were weighed. The average values were calculated. The means for each group were analysed and the foetal placental ratio was calculated. SPSS 17 was used for statistical analysis. RESULTS: There were 44 mice; 15(34%) each in S and SV groups, while Group C had 14(32%) mice who acted as the controls. The mean foetal weight in Group S was 0.65±0.52g which was significantly less (p<0.0001) than1.48±0.19g in Group C. The mean foetal weight in Group SV was 0.97±0.65g which was not significantly different from S (p=0.124). The mean placental weight in Group S was 0.16±0.02g which was significantly less than 0.21±0.05gin Group C (p=0.014). In Group SV it was significantly more than Group S (p<0.0001). The ratio of mean foetal and mean placental weights in the groups C, S and SV were 7.05, 3.92 and4.41 respectively. CONCLUSIONS: Prenatal exposure to tobacco smoke decreased the mean foetal and placental weights and the foetal-placental ratio. This may partly be attributed to oxidative injury induced by free radicals in the tobacco smoke as it is prevented to some extent by simultaneous administration of antioxidants.

The effect of prenatal administration of valproic acid on the survivability and day of hatching of chick embryo.

OBJECTIVE: To assess the effects of prenatal administration of valproic acid on the survivability and day of hatching of chick embryo in comparison with age-matched controls. METHODS: The experimental study was conducted at the Department of Anatomy, Regional Centre of College of Physicians and Surgeons Pakistan, Islamabad, from February 2010 to February 2011. Fertilised chicken eggs were divided into two groups, labelled as experimental group-A and control group-B. Group-A eggs were injected with valproic acid, incubated and hatched. Group-B eggs underwent sham treatment using normal saline. The fully hatched chicks were then evaluated for the day of hatching and survivability, on hatching or on day 22 of incubation whichever was earlier. Outcome was statistically compared with the controls using SPSS 10. RESULTS: The two groups had 30 eggs each. In Group-A 23 (76.66%) chicks hatched out, while there were 7 (23.33%) dead chicks. In Group-B, 28 (93.33%) chicks hatched out and 2 (6.66%) were dead. Chicken embryos exposed to valproic acid in ovo showed increased mortality (p < 0.001) and delayed hatching (p < 0.001). CONCLUSION: Prenatal exposure of chick embryos to valproic acid decreased embryo survival and also delayed hatching compared to age-matched controls.

Wound healing in rat skin subjected to loud noise stress; a light microscopic study.

OBJECTIVE: To determine the effect of loud noise stress on wound healing in a skin tissue. METHODS: The randomised control trial was conducted at the Department of Anatomy, College of Physicians and Surgeons Pakistan Regional Centre, Islamabad, from September 2007 to September 2008. The study comprised 240 male Sprague Dawley rats who were randomised into control group A and experimental group B. Each group comprised 120 animals. Main groups were further subdivided into four subgroups of 30 animals each. After induction of local anaesthesia a linear full thickness skin incision paravertebral to thoracic spine was made. The experimental group B was exposed to loud noise stimulus. The animals were decapitated 1, 3, 5 and 7 days after surgery. Histological data was collected in the incisional space of the wound. Polymorpho-nuclear leukocytes, macrophages, fibroblasts and blood vessels, were analysed quantitatively, whereas re-epithelialisation and content of collagen fibres in the incisional space were analysed qualitatively. RESULTS: Macrophages were decreased initially 3 days after surgery and were increased 5 and 7 days after surgery in the experimental subgroups. Similarly, blood vessels and fibroblast were significantly decreased in experimental subgroups 3 days after surgery, but were increased significantly in experimental subgroups 7 days after surgery. Nothing significant was found regarding re-epithlialisation and collagenisation of wound. CONCLUSION: Loud noise stress affects the important cells involved in the healing of the wound. Therefore, it is expected to have an impact on the stages of wound healing.

Quantitative analysis of macrophages in wound healing of rat skin subjected to loud noise stress.

BACKGROUND: Factors affecting skin wound healing have always been a central consideration in medical practice. Loud noise is biological stressor affecting the body systems at various levels. The present study was taken to study the effect of loud noise stress on the macrophages during wound healing process in male rat skin. METHOD: One hundred and eighty male Sprague Dawley rats were randomly divided into control group-A and experimental group-B. Each group comprised 90 animals. Control and experimental groups were further subdivided into three subgroups of 30 animals each, corresponding to the day of sacrifice of animals, i.e., day 3, 5 and 7 after surgery. After induction of local anaesthesia a linear full thickness incision paravertebral to thoracic spine was made on the dorsum of rat. The experimental group B was exposed to loud noise stimulus (recorded noise of aero planes and gun fire) set at 97dBA to 102 dBA with a sound level meter. The animals were decapitated on day 3, 5 and 7 after surgery. Tissue was processed for paraffin embedding and stained by Hematoxylin and Eosin and Mallory's trichrome stain. Data was collected for the incisional space of the wound. Quantitative data of number of macrophages was analysed by Student's' test for the detection of any significant differences between the mean number in the experimental and control groups. All the quantitative data was expressed as means ± SE. A p-value of ≤ 0.05 was considered statistically significant. RESULTS: In this study macrophages were decreased statistically significantly at day 3 after surgery and thereafter increased significantly on day 5 and 7 after surgery in the experimental subgroups as compared to their match control subgroups. CONCLUSION: These results show that loud noise stress affects the cells (macrophages) involved in the healing of the wound therefore it is expected to have impact on the stages of wound healing.

Ethanol vapour induced dilated cardiomyopathy in chick embryos.

OBJECTIVE: To study the effects of ethanol vapour inhalation on the heart chambers of chick embryo. METHODS: The case-control study was conducted at the College of Physicians and Surgeons Pakistan regional centre in Islamabad from January to October 2007. Both experimental and control groups were divided into three subgroups each, based on the day of the sacrifice. Each group was dissected on day 7, day 10 and day 22 or hatching whichever was earlier. The experimental sub-groups sacrificed on day 7, day 10 and on hatching, were exposed to ethanol vapours till day 6, 9 and 9 of incubation respectively. The diameter of all 4 chambers was measured in experimental hearts and compared with age-matched controls. SPSS 10 was used for statistical analysis. RESULTS: Ethanol vapour exposure caused widening of all heart chambers in the experimental chick embryos sacrifised on day 7 and day 10 compared to the controls. The chambers of newly hatched chick hearts showed dilatation in all the chambers except the left ventricle. CONCLUSION: Ethanol vapour exposure during development affects the heart, resulting in the widening of all heart chambers.The exposure is as dangerous as drinking alcohol. Alcohol vapour exposure during development leads to progressive dilatation in different heart chambers, producing dilated cardiomyopathy.

Effects of immobilisation and re-mobilisation on superficial zone of articular cartilage of patella in rats.

OBJECTIVE: To determine the staining grades and morphological changes in the cells of articular cartilage of patella on immobilisation and re-mobilisation in rats. METHODS: A total of 120 Sprague Dawley rats were divided into four groups of 30 animals. The study was done between July and December 2009. Group I consisted of control animals that were not immobilised. Group 2 were immobilised for four weeks. Group 3 consisted of animals that were immobilised for four weeks and re-mobilised for four weeks. Group 4 consisted of animals that were immobilised for four weeks and re-mobilised for eight weeks. At the end of the period, the knee joint was dissected in sagittal plane along with patella. Tissue specimens were stored in 10% formalin for 48 hours. After processing for making paraffin blocks, 10 microm and 7 microm sections were cut from the same block and stained with Alcian Blue and Haematoxylin and Eosin stain. RESULTS: Extensive necrotic changes were observed on four weeks immobilisation. On four weeks remobilisation after four weeks of immobilization, the superficial zone was sloughed off, but on eight weeks remobilisation after four weeks of immobilization, regeneration was seen in the superficial zone. The superficial zone was affected both in immobilisation and re-mobilisation. CONCLUSION: After eight weeks re-mobilisation, regeneration was still going on. No conclusion can be drawn regarding the exact time required for complete reversibility of the changes in the cartilage.

Teratogenic effects of ethanol vapour exposure on chick embryos.

OBJECTIVE: To observe the effect of ethanol vapours on chick embryos regarding developmental defects and hatchability characteristics. METHODS: An experimental study was performed in the Department of Anatomy at the Regional Center of College of Physicians and Surgeons, Islamabad, from February, 2006 to February, 2007. Chicken eggs after having been exposed to ethanol vapours produced in a specially designed glass chamber, were dissected on day 7, day 10 and day 22 or on hatching and compared with age-matched controls. A breathalyzer was used for monitoring level of ethanol vapours inside the incubator. RESULTS: The results show that experimental group had comparatively more cases of delayed and assisted hatchings as well as growth retardation resulting into failure of retraction of yolk sac, as compared to the controls. CONCLUSION: Ethanol vapour exposure increases the risks of developmental defects with increasing embryonic age. Increased duration of exposure, causes delayed hatching and more assisted hatchings. Newly hatched alcohol exposed chicks showed diminished locomotor activity and poor balance.

A comparative histological study of the surface epithelium and high endothelial venules in the subepithelial compartments of human nasopharyngeal and palatine tonsils.

OBJECTIVE: To compare the thickness and organization of surface epithelium, and high endothelial venules in subepithelial compartments of human nasopharyngeal and palatine tonsils, with reference to functional differences. STUDY DESIGN: Comparative cross-sectional. PLACE AND DURATION OF STUDY: The Anatomy Department of CPSP Regional Centre, Islamabad, from January to December 2005. METHODOLOGY: Thirty samples each of human nasopharyngeal, right palatine and left palatine tonsils were collected by convenience sampling technique. Haematoxylin and eosin stained paraffin sections were examined for surface epithelium. Thickness of stratified squamous and pseudostratified ciliated columnar epithelium was measured, while organization was observed in case of stratified squamous. The high endothelial venules in subepithelial lymphoid compartments were counted. RESULTS: The surface epithelium of nasopharyngeal tonsils (stratified squamous and pseudostratified columnar inclusive) was 63.21+1.93 microm, and that of palatine (stratified squamous) was 143.99+5.94 microm thick (p<0.001). The mean count of high endothelial venules in subepithelial compartments of nasopharyngeal was 1.15+/-0.06 and that of palatine tonsil was 0.93+0.08 (p=0.042). Organization of stratified squamous epithelium was poor in 26 out of 30 nasopharyngeal, and well in all palatine tonsils (p<0.001). CONCLUSION: The surface epithelium of nasopharyngeal tonsil being thinner and poorly organized than that of palatine tonsil might act as a less effective barrier between the antigenic stimulus and subepithelial lymphoid compartments. This may contribute towards higher level of immune response by these compartments of the former, which is endorsed by higher number of high endothelial venules as compared to the latter.

Derangement of chick embryo retinal differentiation caused by radiofrequency electromagnetic fields.

The possible adverse effects of radiofrequency electromagnetic fields (EMF) emitted from mobile phones present a major public concern. Biological electrical activities of the human body are vulnerable to interference from oscillatory aspects of EMF, which affect fundamental cellular activities, in particular, the highly active development process of embryos. Some studies highlight the possible health hazards of EMF, while others contest the hypothesis of biological impact of EMF. The present study was designed to observe the histomorphological effects of EMF emitted by a mobile phone on the retinae of developing chicken embryos. Fertilized chicken eggs were exposed to a ringing mobile set on silent tone placed in the incubator at different ages of development. After exposure for the scheduled duration the retinae of the embryos were dissected out and processed for histological examination. The control and experimental embryos were statistically compared for retinal thickness and epithelial pigmentation grades. Contrasting effects of EMF on the retinal histomorphology were noticed, depending on the duration of exposure. The embryos exposed for 10 post-incubation days exhibited decreased retinal growth and mild pigmentation of the epithelium. Growth retardation reallocated to growth enhancement on increasing EMF exposure for 15 post-incubation days, with a shift of pigmentation grade from mild to intense. We conclude that EMF emitted by a mobile phone cause derangement of chicken embryo retinal differentiation.

Dose related shifts in the developmental progress of chick embryos exposed to mobile phone induced electromagnetic fields.

BACKGROUND: The possible adverse effects of Electromagnetic Fields (EMFs) emitted from mobile phones present a major public concern today. Some studies indicate EMFs effects on genes, free radical production, immunological and carcinogenic effects. On the other hand there are studies which do not support the hypothesis of any biological impacts of EMFs. This study was designed to observe the effects of mobile phone induced EMFs on survival and general growth and development of chick embryo, investigating dose-response relationship if any. METHODS: This was an experimental study in which developing chick embryos were exposed to different doses of mobile phone induced EMFs. For this purpose a mobile phone was placed in the incubator in the centre of fertilised eggs in silent ringing mode and was 'rung' upon from any other line or cell phone. After incubation for 10 or 15 days the eggs were opened and the developmental mile-stones of the surviving embryos were compared with the non exposed subgroup. RESULTS: EMFs exposure significantly decreased the survivability of the chick embryos. The lower doses of EMFs caused growth retardation. However, this effect of growth retardation reallocated to partial growth enhancement on increasing the dose of EMFs and shifted over to definite growth enhancement on further raising the dose. CONCLUSION: There is an adverse effect of EMFs exposure on embryo survivability. Chick embryos developmental process is influenced by EMFs. However, these effects are variable depending upon the dose of EMFs exposure.