Norzana Abd Ghafar

Effects of edible bird's nest (EBN) on cultured rabbit corneal keratocytes

BackgroundThere has been no effective treatment or agent that is available for corneal injury in promoting corneal wound healing. Previous studies on edible birds nest extract (EBN) had reported the presence of hormone-like substance; avian epidermal growth factor that could stimulate cell division and enhance regeneration. This study aimed to investigate the effects of EBN on corneal keratocytes proliferative capacity and phenotypical changes.MethodsCorneal keratocytes from six New Zealand White Rabbits were isolated and cultured until Passage 1. The proliferative effects of EBN on corneal keratocytes were determined by MTT assay in serum-containing medium (FDS) and serum-free medium (FD). Keratocytes phenotypical changes were morphologically assessed and gene expression of aldehyde dehydrogenase (ALDH), collagen type 1 and lumican were determined through RT-PCR.ResultsThe highest cell proliferation was observed when both media were supplemented with 0.05% and 0.1% EBN. Cell proliferation was also consistently higher in FDS compared to FD. Both phase contrast micrographs and gene expression analysis confirmed the corneal keratocytes retained their phenotypes with the addition of EBN.ConclusionsThese results suggested that low concentration of EBN could synergistically induce cell proliferation, especially in serum-containing medium. This could be a novel breakthrough as both cell proliferation and functional maintenance are important during corneal wound healing. The in vitro test is considered as a crucial first step for nutri-pharmaceutical formation of EBN-based eye drops before in vivo application.

The effects of acacia honey on in vitro corneal abrasion wound healing model

BackgroundAcacia honey (AH) has been proven to improve skin wound healing, but its therapeutic effects on corneal epithelium has not been elucidated to date. This study aimed to investigate the effects of AH on cultured corneal epithelial cells (CEC) on in vitro corneal abrasion wound healing model. Six New Zealand white rabbits’ CEC were isolated and cultured until passage 1. Circular wound area was created onto a confluent monolayer CEC using a corneal trephine which mimicked corneal abrasion and treated with 0.025% AH supplemented in basal medium (BM) and complete cornea medium (CCM). Wound healing was measured as the percentage of wound closure by the migration of CEC on day 0, day 3 and day 6, post wound creation. The morphological changes of CEC were assessed via phase contrast microscopy. Gene and protein expressions of cytokeratin (CK3), fibronectin and cluster of differentiation 44 (CD44) in AH treated groups and control groups were determined by real-time PCR and immunocytochemistry, respectively.ResultsCultured CEC exhibited similar morphology of polygonal shaped cells in all culture media. CEC cultured in AH-supplemented media showed higher percentage of wound closure compared to the controls. Gene expression of CK3 increased in AH-supplemented groups throughout the study. Fibronectin expression was increased at the initial stage while CD44 expression was increased at day 3, post wound creation. The protein expression of CEC cultured in all media was in accordance to their respective gene expressions.ConclusionSupplementation of AH in BM and CCM media accelerates CEC wound closure of the in vitro corneal abrasion model by increasing the expression of genes and proteins associated with CEC wound healing.

Effect of acacia honey on cultured rabbit corneal keratocytes.

BackgroundAcacia honey is a natural product which has proven to have therapeutic effects on skin wound healing, but its potential healing effects in corneal wound healing have not been studied. This study aimed to explore the effects of Acacia honey (AH) on corneal keratocytes morphology, proliferative capacity, cell cycle, gene and protein analyses. Keratocytes from the corneal stroma of six New Zealand white rabbits were isolated and cultured until passage 1. The optimal dose of AH in the basal medium (FD) and medium containing serum (FDS) for keratocytes proliferation was identified using MTT assay. The morphological changes, gene and protein expressions of aldehyde dehydrogenase (ALDH), marker for quiescent keratocytes and vimentin, marker for fibroblasts were detected using q-RTPCR and immunocytochemistry respectively. Flowcytometry was performed to evaluate the cell cycle analysis of corneal keratocytes.ResultsCultured keratocytes supplemented with AH showed no morphological changes compared to control. Keratocytes cultured in FD and FDS media supplemented with 0.025% AH showed optimal proliferative potential compared with FD and FDS media, respectively. Gene expressions of ADLH and vimentin were increased in keratocytes cultured with AH enriched media. All proteins were expressed in keratocytes cultured in all media in accordance to the gene expression findings. No chromosomal changes were detected in keratocytes in AH enriched media.ConclusionCorneal keratocytes cultured in media supplemented with 0.025% AH showed an increase in proliferative capacity while retaining their morphology, gene and protein expressions with normal cell cycle. The results of the present study show promising role of AH role in accelerating the initial stage of corneal wound healing.

Anterior fibulocalcaneus muscle of the leg: the mystery deepens.

We read with much interest the article titled ‘‘An Anterior Fibulocalcaneus Muscle: An Anomalous Muscle Discovered in the Anterior Compartment of the Leg’’ (Lambert and Atsas, 2010). We take this excellent opportunity to share our scientific views on the published article. Indeed, the presence of the fibulocalcaneus muscle remains a mystery regarding its evolution and incidence. Interestingly, the present anomaly was observed on both sides of the same cadaver which clearly denotes a developmental defect. The authors also mentioned that there was ‘‘doubling muscular belly and the insertion of the tendon of the fibularis tertius on the right leg’’ (Fig. 2). However, the presence of any other anomalous muscles was not reported. Unfortunately, the nerve supply to the anomalous muscle was never mentioned. This could elucidate the muscular compartment of the anomalous muscle based on its innervation. According to the standard textbook of Anatomy, the anterior compartment muscles of the leg are innervated by the deep peroneal nerve (Standring, 2008). It is a well accepted fact that the anterior compartment of the leg is defined by its anatomical boundaries, not by the innervation. All muscles of the anterior compartment are innervated only by the deep fibular (peroneal) nerve. Hence, the innervation pattern of any anomalous muscle is important. The authors refer to the fibulocalcaneus internus muscle (of MacAlister) and its course posterior to the lateral malleolus. The presence of this muscle was noted to cause posterior ankle pain and impingement as well as tarsal tunnel syndrome (Mellado et al., 1997). The course of the anterior fibulocalcaneus muscle anterior to the lateral malleolus has immense clinical significance. As the tendon passed anterior to the peroneal trochlea, there is a possibility that the calcaneal surface may have provided lot of friction while the tendon moved on its anterosuperior surface. In that case, a detailed observation of the calcaneal surface near the insertion of the muscle may prove to be beneficial. The medial malleolus is more clinically important because of the possibility of posterior ankle pain and impingement or tarsal syndrome. The presence of an extra muscle close to the lateral malleolus may alter the movements and kinematics at surrounding joints. We speculate that the presence of this anomalous muscle may increase the dorsiflexion component if it was proven to be an anterior compartment muscle. To date, there are no literature reports on the clinical implications of these anomalous muscles but these muscles may produce clinical symptoms once they are injured. This interesting article opens the door for further discussion in terms of evolution and clinical implications. We applaud the meticulous work of the authors and thank the Editor for publishing such important case report that enriches the mind of all researchers, anatomists, and clinicians.

Role of MicroRNA in proliferation phase of wound healing

Wound healing is a complex biological process that is generally composed of four phases: hemostasis, inflammation, proliferation, and remodeling. The proliferation phase is crucial for effective healing compared to other phases. Many critical events occur during this phase, i.e., migration of fibroblasts, re-epithelialization, angiogenesis and wound contraction. Chronic wounds are common and are considered a major public health problem. Therefore, there is the increasing need to discover new therapeutic strategies. MicroRNA (miRNA) research in the field of wound healing is in its early phase, but the knowledge of the recent discoveries is essential for developing effective therapies for the treatment of chronic wounds. In this review, we focused on recently discovered miRNAs which are involved in the proliferation phase of wound healing in the past few years and their role in wound healing.

What is the demarcating line between anatomy and exhibitionism

We enjoyed reading the article ‘Exhibitions of human bodies: popular and controversial’ by Mirela et al. [1]. Anatomy uses cadavers for student teaching. There are many institutions all over the world which never have the facility of cadaveric teaching for undergraduate medical students. This has made commercial manufacturers to resort to newer models, charts, 3-D Wgures, primal pictures, software graphics in order to simulate normal human body. There is no reason to believe why those institutions which are capable enough to acquire cadavers should resort to other methods for display or student teaching. Embalming and plastination are meant to preserve the cadaver. The cadaver is preserved under ethical considerations. Do we Wnd any free accessible photo which shows the cadaver being embalmed? Why so much hype for plastination? It is because it can help us to preserve the specimen for longer period. Plastinated specimens are not meant to be displayed in public for commercial use. The authors are very correct in their arguement. Do we allow anyone to take a photograph of the cadaver and display it freely on the website or in public? To display a part of the human body is not only considered unethical but also it can amount to exhibitionism. May we highlight the recent popularity of teaching anatomy by body painting. All over the world many anatomy teachers are resorting to body painting because it has been considered as a modern approach for teaching and learning anatomy. The non medical people take the liberty of body painting in a diVerent manner. Some eminent personalities Wnd it great to paint their bodies and get photographed. Some even shed oV their clothes to paint the body. Can we encourage someone from our anatomy fraternity to do such? What is the diVerence between cadaveric teaching and commercial exposure of one’s body? The obvious questions asked are: (1) Did one get prior permission from the next of the kin of the cadaver to make a plastinated specimen for display? (2) Plastinated specimens are involved with buying and purchasing, so why to promote such publicly from commercial angle? (3) Is it acceptable that one circulates plastinated or human body photographs on the media,T shirts, cups etc.? and (4) Are these plastinated specimens meant for open viewing for general public or it is exclusively meant for medical personnel? There is a decency and religious factor involved in many countries. All human beings look good with their skin on the superWcial surface, not with their skin peeled oV. Why to advertise any human body or its parts by showing such scary photographs? Even in a feature Wlm such is being exhibited with censor warning. Do we really need to draw the attention of public? We agree with the authors that some people do pray in front of the cadavers. How many of the voluntary donations in any country have the fact clearly laid down in the body donation form that part of the deceased body after death would be plastinated and sold outside? The same question was raised by Mirela et al. [1]. The authors mention about the sagittal section of male and female pelvis joined in a sexual relationship [1]. We believe that this is not anatomy rather it is vulgarism. We believe that anatomy teachers and instructors cannot do such. It is only the individuals who want to draw the attention of general people viewing such models. The Wrst and the foremost thing we teach to our students is that they should respect the dead. The authors have rightly pointed it S. Das (&) · N. A. Ghafar Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abd Aziz, 53000 Kuala Lumpur, Malaysia e-mail: das_srijit23@rediffmail.com

Identifying Potential Therapeutics for Osteoporosis by Exploiting the Relationship between Mevalonate Pathway and Bone Metabolism

BACKGROUND Osteoporosis is a silent skeletal disease characterized by low bone mass and destruction of skeletal microarchitecture, leading to an increased fracture risk. This occurs due to an imbalance in bone remodelling, whereby the rate of bone resorption is greater than bone formation. Mevalonate pathway, previously known to involve in cholesterol synthesis, is an important regulatory pathway for bone remodelling. OBJECTIVE This review aimed to provide an overview of the relationship between mevalonate pathway and bone metabolism, as well as agents which act through this pathway to achieve their therapeutic potential. DISCUSSION Mevalonate pathway produces farnesyl pyrophosphate and geranylgeranyl pyrophosphate essential in protein prenylation. An increase in protein prenylation favours bone resorption over bone formation. Non-nitrogen containing bisphosphonates inhibit farnesyl diphosphate synthase which produces farnesyl pyrophosphate. They are used as the first line therapy for osteoporosis. Statins, a well-known class of cholesterol-lowering agents, inhibit 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, the rate-determining enzyme in the mevalonate pathway. It was shown to increase bone mineral density and prevent fracture in humans. Tocotrienol is a group of vitamin E commonly found in palm oil, rice bran and annatto bean. It causes degradation of HMG-CoA reductase. Many studies demonstrated that tocotrienol prevented bone loss in animal studies but its efficacy has not been tested in humans. CONCLUSION Mevalonate pathway can be exploited to develop effective antiosteoporosis agents.

Annatto-derived tocotrienol stimulates osteogenic activity in preosteoblastic MC3T3-E1 cells: a temporal sequential study

Purpose Annatto-derived tocotrienol (AnTT) has been shown to improve bone formation in animal models of osteoporosis. However, detailed studies of the effects of AnTT on preosteoblastic cells were limited. This study was conducted to investigate the osteogenic effect of AnTT on preosteoblast MC3T3-E1 cells in a time-dependent manner. Materials and methods Murine MC3T3-E1 preosteoblastic cells were cultured in the different concentrations of AnTT (0.001–1 µg/mL) up to 24 days. Expression of osteoblastic differentiation markers was measured by qPCR (osterix [OSX], collagen 1 alpha 1 [COL1α1], alkaline phosphatase [ALP], and osteocalcin [OCN]) and by fluorometric assay for ALP activity. Detection of collagen and mineralized nodules was done via Direct Red staining and Alizarin Red staining, respectively. Results The results showed that osteoblastic differentiation-related genes, such as OSX, COL1α1, ALP, and OCN, were significantly increased in the AnTT-treated groups compared to the vehicle group in a time-dependent manner (P<0.05). Type 1 collagen level was increased from day 3 to day 15 in the AnTT-treated groups, while ALP activity was increased from day 9 to day 21 in the AnTT-treated groups (P<0.05). Enhanced mineralization was observed in the AnTT-treated groups via increasing Alizarin Red staining from day 3 to day 21 (P<0.05). Conclusion Our results suggest that AnTT enhances the osteogenic activity by promoting the bone formation-related genes and proteins in a temporal and sequential manner.

Acanthamoeba-mediated cytopathic effect correlates with MBP and AhLBP mRNA expression

BackgroundIn recent years, the concern of Acanthamoeba keratitis has increased since the infection is often associated with contact lens use. Partial 18S rRNA genotypic identification of Acanthamoeba isolates is important to correlate with pathophysiological properties in order to evaluate the degree of virulence. This is the first report of genotypic identification for clinical isolates of Acanthamoeba from corneal scrapings of keratitis in Malaysia. This study is also the first to correlate the mRNA expression of MBP and AhLBP as virulent markers for axenic strains of Acanthamoeba.ResultsIn this study, ten clinical isolates were obtained from corneal scrapings. Rns genotype and intra-genotypic variation at the DF3 region of the isolates were identified. Results revealed that all clinical isolates belonged to the T4 genotype, with T4/6 (4 isolates), T4/2 (3 isolates), T4/16 (2 isolates) and one new genotype T4 sequence (T4/36), being determined. The axenic clinical isolates were cytopathogenic to rabbit corneal fibroblasts. MBP and AhLBP mRNA expression are directly correlated to Acanthamoeba cytopathic effect.ConclusionsAll ten Malaysian clinical isolates were identified as genotype T4 which is predominantly associated with AK. Measuring the mRNA expression of Acanthamoeba virulent markers could be useful in the understanding of the pathogenesis of Acanthamoeba keratitis.

Ankle injuries in sports: anatomical considerations and clinical implications

Ankle injuries are commonly seen in various sports. The main aim of the present review was to highlight the normal anatomy of the ankle, mechanism of injuries related to sports, congenital abnormalities or anatomical variations related to ankle injury and discuss its effective management. A review of literature was done to determine the ankle injuries which occur as a result of various sports related activities. We documented all sports which involved injury to the ankle joint. The anatomy of various structures in the sole of foot and their involvement in injuries were discussed at length. The anatomical knowledge of ankle injury may be beneficial for future diagnosis and treatment purpose.