Protocol paper: kainic acid excitotoxicity-induced spinal cord injury paraplegia in Sprague-Dawley rats

BACKGROUND: Excitotoxicity-induced in vivo injury models are vital to reflect the pathophysiological features of acute spinal cord injury (SCI) in humans. The duration and concentration of chemical treatment controls the extent of neuronal cell damage. The extent of injury is explained in relation to locomotor and behavioural activity. Several SCI in vivo methods have been reported and studied extensively, particularly contusion, compression, and transection models. These models depict similar pathophysiology to that in humans but are extremely expensive (contusion) and require expertise (compression). Chemical excitotoxicity-induced SCI models are simple and easy while producing similar clinical manifestations. The kainic acid (KA) excitotoxicity model is a convenient, low-cost, and highly reproducible animal model of SCI in the laboratory. The basic impactor approximately cost between 10,000 and 20,000 USD, while the kainic acid only cost between 300 and 500 USD, which is quite cheap as compared to traditional SCI method. METHODS: In this study, 0.05 mM KA was administered at dose of 10 µL/100 g body weight, at a rate of 10 µL/min, to induce spinal injury by intra-spinal injection between the T12 and T13 thoracic vertebrae. In this protocol, detailed description of a dorsal laminectomy was explained to expose the spinal cord, following intra-spinal kainic acid administration at desired location. The dose, rate and technique to administer kainic acid were explained extensively to reflect a successful paraplegia and spinal cord injury in rats. The postoperative care and complication post injury of paraplegic laboratory animals were also explained, and necessary requirements to overcome these complications were also described to help researcher. RESULTS: This injury model produced impaired hind limb locomotor function with mild seizure. Hence this protocol will help researchers to induce spinal cord injury in laboratories at extremely low cost and also will help to determine the necessary supplies, methods for producing SCI in rats and treatments designed to mitigate post-injury impairment. CONCLUSIONS: Kainic acid intra-spinal injection at the concentration of 0.05 mM, and rate 10 µL/min, is an effective method create spinal injury in rats, however more potent concentrations of kainic acid need to be studied in order to create severe spinal injuries.

Construction of a doxycycline inducible lentivirus that expresses stem cell-specific miR-302 cluster

@#Introduction: The polycistronic miR-302 cluster encodes five miRNA genes that have an important role in the regulation of embryonic stem cell function. Studies showed that the miR-302 cluster can reprogram both mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) with high efficiency. The aim of this study was to generate an inducible lentivirus that expresses miR-302 cluster in order to further investigate somatic cell reprogramming by these miRNAs. Materials and Methods: The miR-302 cluster was amplified by polymerase chain reaction technique from human genomic DNA and was ligated into pTRIPz, an inducible lentiviral vector. Results: MRC5 fibroblasts and HEK293 (human embryonic kidney) cells were infected with pTRIPz-302 cluster lentivirus and the family of 302 miRNAs were strongly expressed in HEK293 cells but lowly expressed in MRC5 fibroblasts. When cultured in hESC conditions, MRC5 cells expressed only low levels of DNMT3B, Nanog, Oct4 and Lin28 and failed to show stem cell induction. The red fluorescent expression seen in the majority of MRC5 cells, indicated that the rate of infection by lentivirus was efficient. Discussion: The efficiency of reprogramming may be improved perhaps by either using a different cell type or a high expression vector with a different type of promoter.

CNS germinoma with extensive calcification: An unusual histologic finding

@#Introduction: Intratumoral calcification is a feature that is more often observed in pineal parenchymal tumour than germinoma. We describe a 13-year-old male with pineal region germinoma demonstrating extensive intratumoral calcification. Case report: He presented with worsening headache that was associated with fatigue, nausea and vomiting. Radiologic examination revealed a multilobular mass in the pineal region with internal calcifications. Biopsy showed a pure germinoma with unusually extensive calcification. Discussion: Although a diagnosis may be suggested with a careful evaluation of imaging, there is no pathognomonic pattern. Thus, histologic verification is necessary for most pineal region masses.

Mesenchymal Stromal Cells from the Maternal Segment of Human Umbilical Cord is Ideal for Bone Regeneration in Allogenic Setting

Umbilical cord (UC) is a discarded product from the operating theatre and a ready source of mesenchymal stromal cells (MSCs). MSCs from UC express both embryonic and adult mesenchymal stem cell markers and are known to be hypoimmunogenic and non-tumorigenic and thus suitable for allogeneic cell transplantation. Our study aimed to determine the degree of immunotolerance and bone-forming capacity of osteodifferentiated human Wharton's jelly-derived mesenchymal stromal cells (hWJ-MSCs) from different segments of UC in an allogenic setting. UCs were obtained from healthy donors delivering a full-term infant by elective Caesarean section. hWJ-MSCs were isolated from 3 cm length segment from the maternal and foetal ends of UCs. Three-dimensional fibrin constructs were formed and implanted intramuscularly into immunocompetent mice. The mice were implanted with 1) fibrin construct with maternal hWJ-MSCs, 2) fibrin construct with foetal hWJ-MSCs, or 3) fibrin without cells; the control group received sham surgery. After 1 month, the lymphoid organs were analysed to determine the degree of immune rejection and bone constructs were analysed to determine the amount of bone formed. A pronounced immune reaction was noted in the fibrin group. The maternal segment constructs demonstrated greater osteogenesis than the foetal segment constructs. Both maternal and foetal segment constructs caused minimal immune reaction and thus appear to be safe for allogeneic bone transplant. The suppression of inflammation may be a result of increased anti-inflammatory cytokine production mediated by the hWJ-MSC. In summary, this study demonstrates the feasibility of using bone constructs derived from hWJ-MSCs in an allogenic setting.

Undergraduate medical education research in Malaysia: time for a change

Objective: Special Study Module [SSM] is a mandatory research module implemented in Universiti Kebangsaan Malaysia [UKM]. The objective of this paper is to provide a brief overview on the student research activities and to find out the outcome measures in terms of publication Methods: It was a retrospective study done on SSM research projects at UKM. The SSM research is conducted from beginning of year-4 until 1st seven weeks of year-5. In year-4, students are assigned to a facultysupervisor in small groups and spend every Thursday afternoon to plan and carry the research. Whole first seven weeks of year-5, students are placed with their supervisor continuously to collect data, do analysis, write report and present in the scientific conference. Outcomes of 5-years SSM research-projects starting from 2008/2009 to 2012/2013 academic session were analyzed. Results: Total 257 projects were completed and presented in annual scientific meetings from which 57 [22.2%] articles were published in peer reviewed journals. Conclusion: Mandatory undergraduate student research project brings an opportunity to develop students' capacity building from conception to final report writing and thereby narrowing the gap between education and practice. Medical schools should implement research module to bring changes in research and publication culture of undergraduate medical education

Human telomerase reverse transcriptase expression in ovarian tumors.

Background : Ovarian cancer is the 6 th most common cancer among women. In ovarian tumors, the borderline category is not well defined due to the difficulty in assessing stromal invasion. The World Health Organization (WHO) defined it as tumor that lacks obvious invasion of the stroma with mitotic activity and nuclear abnormalities intermediate between clearly benign and unquestionably malignant. Telomerase is expressed in many human cancers and is hence a potential biomarker for cancer. Immunohistochemical study of anti-human telomerase enzyme reverse transcriptase (hTERT) antibody allows direct visualization of its expression. The aim of this study was to determine the expression of hTERT and serum CA-125 level in ovarian epithelial tumors, and their ability to distinguish borderline tumor from malignancy. Materials and Methods : This was a retrospective study on 68 ovarian epithelial tumors, comprising of 41 cystadenocarcinoma, 22 borderline tumor and five cystadenoma. By immunohistochemistry, hTERT expression was graded as negative (0-10%), focal (11-25%), regional (26-75%) and diffuse (>75%) positivity. Results : hTERT protein expression in ovarian cystadenocarcinoma, borderline tumor and cystadenoma were 71.4%, 59.1% and 0%, respectively. hTERT and CA-125 had a linear relationship with tumor grade and stage. hTERT protein is detected as large granules/speckled in the cytoplasm and nuclei of ovarian tumors. Conclusions : hTERT protein was highly expression in ovarian epithelial carcinoma. However, the difference between carcinoma and borderline tumor was not statistically significant (P-value = 0.51). It is not an independent biomarker to differentiate borderline tumor from malignant tumor. We suggest using the combination of hTERT immunohistochemistry and serum CA-125 to evaluate difficult situations where histological evaluation fails to distinguish malignant from borderline ovarian tumor.

Immunohistochemical study of p16 INK4A and survivin expressions in cervical squamous neoplasm.

Introduction:Cervical cancer is the second most common cancer affecting Malaysian women. Despite the implementation of pap smear screening, many women are still diagnosed only in the advanced stage of cervical cancer. This could partly be due to failure of detection of its precursor lesions; hence the need to search for novel biomarkers to assist in the screening and diagnosis of cervical neoplasia. This study aims to determine the expression of p16INK4A and survivin as possible predictive biomarkers in cervical squamous neoplasm. Material and Methods: This is a retrospective study on 201 cases of cervical neoplasm comprising of 129 cervical intraepithelial neoplasia (CIN) and 72 squamous cell carcinoma (SCC). All samples were evaluated by two independent observers using p16INK4A and survivin monoclonal antibodies. The p16 INK4A expression was graded as negative, focal and diffuse positivity. The intensity for survivin expression was graded as weak, moderate and intense. Results: It is seen that p16 INK4A expression in CIN 1, CIN 2 and CIN 3 were 25.4%, 42.9% and 95.9% respectively. Majority of SCC (98.6%) showed p16 INK4A expression. Survivin expressions in CIN 1, CIN 2, CIN 3 and SCC were 56.7%, 33.4%, 87.5% and 98.6%. There was a linear relationship between increasing grade of CIN and p16 INK4A expressions. Conclusion: Our study showed that p16 INK4A expressions correlate well with the increasing grade of CIN. Although survivin does not correlate well to the increasing grade of CIN, it could be useful in differentiating CIN 3 from SCC.

Utility of Ki-67 and p53 in distinguishing cervical intraepithelial neoplasia 3 from squamous cell carcinoma of the cervix.

The differentiation between cervical intraepithelial neoplasia 3 (CIN 3) and early squamous cell carcinoma (SCC) of the cervix may be difficult in certain situations. Identification of invasion beyond the basement membrane is the gold standard for the diagnosis of the latter. The objective of this study was to determine whether the use of Ki-67 and p53 could help in solving the above dilemma. This was a retrospective study on 61 cases of cervical neoplasms comprising of 25 cases of CIN 3 and 36 SCC. All cases were evaluated by immunohistochemistry using Ki-67 and p53 monoclonal antibodies. Results showed that the differences of Ki-67 and p53 expression between CIN 3 and SCC were statistically significant. In conclusion, Ki-67 and p53 may serve as helpful adjuncts to routinely-stained histological sections in differentiating between CIN 3 and SCC.