Efficient Inhibition of Human Glioma Development by RNA Interference-Mediated Silencing of PAK5
2015

Xuefeng Gu
Institute of Neurology
Guangdong Medical College
ZhanjiangChina

Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases
Affiliated Hospital of Guangdong Medical College
Zhan-jiangChina

Ce Wang
Department of Neurosurgery
Institute of Neurology
The Fourth Affiliated Hospital of Harbin Medical University
HarbinChina

Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases, Affiliated Hospital of Guangdong Medical College
Guangdong Medical College
524001, 524001Zhanjiang, ZhanjiangChina., China

Xuefeng Wang
Guoda Ma
Institute of Neurology
Guangdong Medical College
ZhanjiangChina

Department of Neurosurgery
Institute of Neurology
The Fourth Affiliated Hospital of Harbin Medical University
HarbinChina

Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases, Affiliated Hospital of Guangdong Medical College
Guangdong Medical College
524001, 524001Zhanjiang, ZhanjiangChina., China

You Li
Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases
Affiliated Hospital of Guangdong Medical College
Zhan-jiangChina

Lili Cui
Institute of Neurology
Guangdong Medical College
ZhanjiangChina

Yanyan Chen
Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases
Affiliated Hospital of Guangdong Medical College
Zhan-jiangChina


Keshen Li
Institute of Neurology
Guangdong Medical College
ZhanjiangChina

Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases
Affiliated Hospital of Guangdong Medical College
Zhan-jiangChina

Keshen Li
Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases
Affiliated Hospital of Guangdong Medical College
Zhan-jiangChina

Zhao Keshen Li
Efficient Inhibition of Human Glioma Development by RNA Interference-Mediated Silencing of PAK5

Int. J. Biol. Sci
11201510.7150/ijbs.9193Received: 2014.03.23; Accepted: 2014.12.10;I In nt te er rn na at ti io on na al l J Jo ou ur rn na al l o of f B Bi io ol lo og gi ic ca al l S Sc ci ie en nc ce es sResearch Paper † These authors contributed equally to this work.  Corresponding author: Please see http://ivyspring.com/terms for terms and conditions.gliomaPAK5tumor developmentRNA interferencecell cycle arrest
Glioma is the most common type of primary intracranial tumor and is highly lethal due to its pathogenetic location, high invasiveness, and poor prognosis. Even combined surgery and chemoradiotherapy do not effectively rescue glioma patients. Molecular target therapy is considered a safe and promising therapy for glioma. The identification of a novel, effective target protein in gliomas is of great interest. We found that PAK5 was highly expressed in the tumor tissues of glioma patients and human glioma cell lines. We then used a lentivirus-delivered short hairpin RNA to stably silence PAK5 expression in glioma cells and explore its influence. The results showed that the inhibition of PAK5 reduced cell viability and delayed the cell cycle at the G0/G1 phase in the glioma cells with PAK5 high expression. In addition, silencing PAK5 expression in U87 cells weakened their colony formation ability and in vivo tumorigenesis ability. Further studies demonstrated that PAK5 inhibition led to an increase in cleaved caspase 3 and a decrease in β-catenin. In conclusion, our results suggest that the inhibition of PAK5 by RNA interference might efficiently suppress tumor development of glioma cells with PAK5 high expression. This finding provides a novel, promising therapeutic target for glioma treatment.

Introduction

Glioma is the most common type of primary brain tumor and accounts for approximately 46 percent of intracranial tumors. Its peak incidence occurs at 30-40 years of age. Glioma is the second leading cause of death in cancer patients less than 34 years of age [1]. With an annual incidence of 3-5/100,000 individuals, glioma is an infrequent malignancy, but its pathogenetic location, high invasiveness, and poor prognosis make glioma a highly lethal cancer. In spite of combined therapies of craniotomy, radiotherapy, and chemotherapy, the overall median survival period is approximately 15 months [2]. Effective glioma therapy remains a difficult challenge for medical professionals.

The investigation of the genes that influence glioma development will advance our understanding of the molecular pathogenesis of this deadly disease and, consequently, its treatment. The role of the p21-activated kinase (PAK) family in tumorigenesis has received increasing attention. PAKs are ser-Ivyspring International Publisher ine/threonine kinases that function as downstream effectors in various oncogenic signaling pathways [3]. Members of the PAK family, which includes six isoforms, can be classified into two groups based on their sequences and functions. While the roles of group A PAKs (PAK1-3) in the signaling pathways of nervous system diseases have been thoroughly investigated, the functions of the group B PAKs (PAK4-6) remain unclear [4].

PAK5 expression is particularly high in the brain. In addition, PAK5 is generally expressed in nerve cells but not glial cells [5]. PAK5 can phosphorylate Raf1 at Ser338, which promotes translocation of Raf1 to the mitochondria [6]. In the mitochondria, Raf1 forms a complex with Bcl2 and further phosphorylates the pro-apoptotic protein BAD at the Ser112 site. Although Bcl-2 is an anti-apoptotic factor, the binding of Bcl-2 to BAD induces the release of many pro-apoptotic proteins from the mitochondria and initiates apoptosis. The phosphorylation of BAD at Ser112 eliminates Bcl-2 binding [7]. PAK5 is overexpressed in some colorectal cancers and plays an important role in the migration of colorectal carcinoma cells [8]. PAK5 is also significant for neurite growth in neuroblastoma cells [9]. However, the role of PAK5 in gliomas remains unclear.

In this study, we determined that PAK5 expression is prominently higher in cancerous tissues of glioma patients than in pericancerous tissues. We used lentivirus-mediated short hairpin RNA (shRNA) to efficiently silence PAK5 expression in human glioma cells, which resulted in a decrease in cell viability and cell cycle arrest in the G0/G1 phase in glioma cells with PAK5 high expression. Colony formation and in vivo tumorigenesis were also reduced by PAK5 silencing in these glioma cells. PAK5 inhibition also caused an increase in cleaved caspase 3 and a decrease in β-catenin. Our findings demonstrate that inhibition of PAK5 by lentivirus-mediated RNA interference (RNAi) significantly suppresses tumor development in glioma cells with PAK5 high expression.

Materials and Methods

Tissue Samples

Samples of human glioma tissues and adjacent normal tissues were collected during the surgeries of 40 glioma patients. The pathologic features of the tumors were evaluated by WHO criterion. All patients provided written informed consent. No patients had previously received chemotherapy or radiotherapy. The samples were soaked in RNALater (Qiagen GmbH, Hilden, Germany) for RNA extraction and preserved in 10% neutral-buffered formalin for histopathological and immunohistochemical analyses.

The study was approved by the Ethics Committee of the Affiliated Hospital of Guangdong Medical College, and the Helsinki Declaration of Human Rights was strictly observed.

Immunohistochemistry

Paraffin-embedded tissues were stained with hematoxylin and eosin to analyze morphological changes. The immunohistochemical antibodies were purchased from Abcam (Cambridge, MA, USA). The primary antibody was a PAK5 antibody (ab110069), and the secondary antibody was a goat polyclonal secondary antibody to rabbit IgG (HRP) (ab6721).

Cell Culture

The human glioma cell lines U87, SHG-44, CHG-5, and U251 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). U87, SHG-44 and U251 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO, Gaithersburg, MD) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C under humidified air containing 5% CO2. CHG-5 cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640, GIBCO) under the same culture conditions as above.

PAK5 silencing using lentivirus-delivered shRNA and PAK5 expressing using PAK5 vector Two shRNA candidates with PAK5 target sequences were used: PAK5-shRNA1 (5'-CGGG ATTACCACCATGACAAT-3') and PAK5-shRNA2 (5'-GCTCCTATGAAGACAATCGTT-3'). The sequence of the scrambled shRNA (Scr-shRNA) was 5'-TTCTCCGAACGTGTCACGT-3'. Scr-RNAi was used as a negative RNAi control. The oligonucleotides encoding the shRNA sequences were inserted into the GFP expression vector pGCL-GFP. The recombinant virus was packaged using Lentivector Expression Systems, and U87, SHG-44, and CHG-5 cells were infected. After 3 days, GFP-positive cells were counted under a fluorescence microscope (OLYMPUS, Japan). PAK5 expression after shRNA infection was determined by quantitative real-time PCR (qRT-PCR) or western blotting on the 4th day.

pCMV6-Myc-PAK5WT vector (Plasmid 16019) was purchased from addgene (Addgene Inc., Cambridge, MA, USA). PAK5 expression of infected U87 cells were determined by western blotting 3 days after cell infection.

RNA extraction and qRT-PCR

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA) and from tissues using RNALater according to the manufacturer's in-structions. cDNA was synthesized using a RevertAid First-Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). Gene expression levels were detected by qRT-PCR using a standard SYBR Green RT-PCR Kit (Takara, Kyoto, Japan). The relative levels of the PAK5 gene mRNA transcripts were normalized to the control, GAPDH.

Western blotting

Cell lysates were subjected to SDS-PAGE. The blots were incubated with the desired primary antibodies, which included anti-PAK5 (Abcam, ab110069), anti-cleaved caspase 3, and anti-β-catenin (both from Cell Signaling Technology, Beverly, MA), followed by incubation with an anti-rabbit IgG peroxidase-conjugated secondary antibody (Abcam, ab6721) and chemiluminescent substrates. Hybridization with anti-GAPDH (Cell Signaling Technology) was used to confirm equal protein loading.

Cell viability assay

Cell viability was evaluated by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (Sigma Chemical, St. Louis, MO) assay. The cells of test groups were plated at a final concentration of 3,000 cells/well in 96-well culture plates for different culture times. Then, MTT (10 µl, 5 mg/ml) was added to each well and incubated at 37°C for 1 h. The reaction was terminated by replacing the MTT-containing medium with 100 μl of DMSO, and the formazan salts were dissolved by gentle shaking for approximately 10 min at room temperature. The optical density (OD) of each well was measured at 490 nm using an ELISA reader (ELx808 Bio-Tek Instruments, USA).

Cell cycle assay

Cells were seeded into 6-well culture plates and harvested at 48 hours by centrifugation at 1200 rpm for 5 min. After washing twice with pre-cooled PBS (pH 7.4), the cells were fixed in 70% alcohol. The percentage of cells in each stage of the cell cycle was determined by staining with propidium iodide (PI, Sigma). The cell cycle distribution was analyzed using a flow cytometer (FACSCalibur, BD) in accordance with the manufacturer's guidelines.

Colony formation assay

Exponentially growing cells were suspended in complete growth medium and seeded in 6-well plates at 500 cells/well. The plates were maintained at 37ºC in a humidified incubator with 5% CO 2 for one week. After fixation in paraformaldehyde, the colonies were stained with crystal violet for 10 min, followed by imaging using a digital camera.

Tumorigenesis assay

Male nude mice (7-8 weeks old, weighing 18-20 g) were obtained from the Shanghai Experimental Center, Chinese Science Academy, Shanghai, and maintained at an animal facility under pathogen-free conditions. All animals received humane care according to the National Research Council's guidelines. U87 cells infected with Scr-shRNA or PAK5-shRNA lentivirus were resuspended in PBS at a final density of 1 × 10 7 cells/ml. A 100-μl aliquot of the cell suspension (equivalent to 1 × 10 6 U87 cells) was injected into the axilla of the nude mice in the corresponding group. Some mice were sacrificed 3 weeks after injection, and tumor growth was examined. The other mice were used to detect the time of visible tumors (diameter > 3 cm).

Statistical methods

The data were expressed as the mean ± SEM. Statistical significance was determined using Student's t-test, one-way ANOVA and chi-square test using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA). A value of P < 0.05 was considered statistically significant.

Results

Upregulation of PAK5 expression in gliomas

PAK5 expression levels in human gliomas have not been previously determined, and thus, we first detected PAK5 mRNA expression in freshly sampled gliomas and in matched pericancerous tissues from 40 glioma patients. The data suggested that PAK5 mRNA expression is markedly higher in cancerous tissue than in pericancerous tissue in most (35 to 40) glioma patients (Fig. 1A). To confirm the high expression of PAK5 in gliomas, we performed immunohistochemical analysis to evaluate PAK5 expression in human gliomas. The glioma tissue exhibited strong positive staining by the anti-PAK5 antibody, whereas PAK5 expression levels were low in most pericancerous tissues of glioma patients (Fig. 1B). However, PAK5 expression had no obvious correlations with grades of glioma tumors (Table 1). On the other hand, immunoblot analysis demonstrated that PAK5 expression levels are high in human glioma cell lines, particularly in U87 and SHG44 cells (Fig. 1C). Taken together, these data suggest that PAK5 expression is upregulated in human gliomas.

Lentivirus-delivered targeted RNAi stably inhibits PAK5 expression in human glioma cells

While normal glial cells do not express PAK5 [5], our results indicated that glioma cells express high levels of PAK5. This difference between normal glial cells and glioma cells suggests that PAK5 expression might affect glioma development. To investigate the role of PAK5 in glioma tumorigenesis, we used lenti-virus-mediated RNAi to silence PAK5 expression in a human glioma cell line. Lentiviral vectors expressing PAK5-specific shRNA and Scr-shRNA were used to infect U87 cells. After three days, high-efficiency infection was observed ( Fig. 2A). To determine the effectiveness of the inhibition of PAK5 expression by the PAK5-shRNA-expressing lentiviral vectors, we detected PAK5 mRNA and protein levels in infected cells by real time-PCR and western blot. Both PAK5-shRNAs significantly reduced PAK5 mRNA expression (Fig. 2B). In addition, PAK5 protein levels were also prominently decreased in the two PAK5-shRNA-infected groups compared with the Scr-shRNA infected group (Fig. 2C). In addition, SHG-44 and CHG-5 cells also showed the similar results ( Fig. 2D and E). Therefore, both types of PAK5-shRNA efficiently downregulated PAK5 expression in human glioma cells.

Inhibition of PAK5 significantly augments tumor growth in glioma cells with PAK5 high expression

We next explored the effects of PAK5 inhibition in glioma cells. First, we used a MTT assay to assess cell growth dynamics in Scr-shRNA-and PAK5-shRNA-infected cells. After 5 days, in U87 and SHG-44 cells, PAK5-shRNA-infected cells displayed remarkable cell growth inhibition compared with Scr-shRNA-infected cells. At day 5, the cell viability of the Scr-shRNA-infected group was 4 times higher than that of the PAK5-shRNA-infected groups ( Fig. 3A and B). However, there was no significant difference between Scr-shRNA-and PAK5-shRNA-infected CHG-5 cells, cells with low PAK5 expression (Fig. 3C). To further determine the effect of PAK5 in glioma cells with high PAK5 expression, we enhanced PAK5 expression by transfecting PAK5 vector in PAK5-shRNA1-infected U87 cells (Fig. 3D). The results showed that enhancing PAK5 expression rescued the decrease of cell viability resulting from RNAi-mediated PAK5 knockdown (Fig. 3E).

Flow cytometric analysis of cell cycle of U87 cells by PI staining revealed that PAK5 inhibition resulted in an increase in cells detained at the G0/G1 phase and a decrease in cells at the G2/M phases (Fig. 4B). These data demonstrate that PAK5 inhibition forces U87 cells into the quiescent phase. Moreover, the colony-forming ability of the PAK5-shRNA infected groups also obviously decreased, in contrast to the Scr-shRNA infected group  (Fig. 4A). These data demonstrate that the inhibition of PAK5 expression decreases U87 cell growth in vitro. A nude mouse tumorigenesis test revealed that the tumorigenesis ability of U87 cells was also weakened by silencing of PAK5 expression (Fig. 4C and D). Taken together, these results suggest that the inhibition of PAK5 expression suppresses tumor growth in glioma cells with PAK5 high expression.

PAK5 silencing causes caspase 3 activation and the downregulation of β-catenin

Next, we examined the mechanism by which PAK5 inhibition suppresses glioma growth. Role of PAK5 in tumor development has been poorly described, and some studies have reported that PAK5 has an anti-apoptotic function that is dependent on AKT activation [7,10]. Therefore, we primarily focused on AKT-associated signaling pathways. Western blot analysis revealed that cleaved caspase 3 levels were higher in PAK5-shRNA-infected cells than in Scr-shRNA-infected cells (Fig 5), indicating that PAK5 silencing promotes cell apoptosis in glioma cells. However, our above results demonstrated that PAK inhibition results in cell cycle arrest in the G0/G1 phase (Fig. 4A). Therefore, we also analyzed the levels of proteins in the AKT-associated cell cycle pathway, which revealed that β-catenin protein levels were significantly reduced by PAK5 inhibition in glioma cells (Fig. 5). β-catenin might promote the expression of cyclin D1, thereby contributing to cell cycle progression [11]. The decrease in the β-catenin level may lead to cell cycle arrest in glioma cells. Moreover, enhancing PAK5 expression reverse this trend (Fig 5). These results demonstrated that silence of PAK5 silencing activated caspase 3 and downregulated β-catenin in the glioma cells.

Discussion

Gliomas are the most malignant brain tumors, and glioma patients do not survive more than a few months after diagnosis, even when treated with a combination of surgery, radiotherapy, and chemotherapy. As the in-depth understanding of the molecular mechanisms of glioma development has improved, molecular targeted therapy has been proposed for the treatment of gliomas. EGFR [12], PTEN [13], and PDGFRα [14] have important roles in glioma development and have been identified as protein targets for glioma treatment. However, therapies that target these proteins and their associated pathways do not completely cure glioma [15], and thus, novel therapeutic targets are urgently needed.

In the present study, high PAK5 expression was observed in glioma tissue from glioma compared to adjacent normal tissues. The in vitro study also revealed that PAK5 was expressed at high levels in most human glioma cell lines. We used lentivirus-delivered specific shRNAs to efficiently silence PAK5 expression in human glioma cells. Inhibition of PAK5 significantly resulted in the decrease of cell viability in the glioma cells with PAK5 high expression. Moreover, PAK5 inhibition led to cell cycle arrest in the G0/G1 phase in U87 cells. In addition, U87 cells infected with PAK5-shRNAs had prominently reduced colony formation and in vivo tumorigenesis ability. Further examination revealed that silencing PAK5 resulted in an increase in cleaved caspase 3 levels and a decrease in β-catenin levels in U87 cells. These re-sults suggest that inhibition of PAK5 by lentivirus-mediated RNAi efficiently suppresses tumor development in the glioma cells with PAK5 high expression.

In the brain, PAK5 is constitutively expressed in nerve cells but not in glial cells [5]. However, our data revealed high PAK5 expression levels in glioma tissue but no detectable expression in the matched pericancerous tissues. Thus, PAK5 may play an important role in glioma development. PAK5 is one of the most poorly understood PAK family isoforms. Previous studies have reported a role for PAK5 in inhibiting cell apoptosis [7,16], and PAK5 overexpression promotes neurite growth in a neuroblastoma cell line [9]. In the present study, we determined that silencing PAK5 increased cell apoptosis in glioma cells. The AKT pathway plays an important role in the PAK5-mediated anti-apoptotic pathway [6]. In addition, Mdm2, which is a downstream gene of AKT, is highly expressed in malignant human glioma cells [17]. However, we determined that PAK5 inhibition had no impact on the level of the anti-apoptotic protein Bcl-2, which is upregulated by activation of the AKT/Mdm2 pathway (data not shown). By contrast, our results demonstrated that PAK5 inhibition restrained the cell cycle at the G0/G1 phases. Han ZX, et al. also demonstrated that suppression of PAK5 in human glioma cell lines led to G1 phase arrest and the increase of cell apoptosis [18]. The mechanism underlying this effect may involve the decrease in β-catenin induced by PAK5 inhibition (Fig. 4). β-catenin is upregulated by the AKT/GSK3β path-way, which thereby triggers the cell cycle by enhancing cyclin D1 expression [11]. Additionally, the study of Han ZX et al. also showed that PAK 5 played an important role in cell migration and invasion potentially through PAK5-Egr1-MMP2 signaling pathway [18]. Notably, inhibition of PAK5 had no obviously effect on the glioma cell with PAK5 low expression (Fig. 3C). The reason may be that these cells have been screening during the process of tumor development and these screened cells have already activated some compensatory PAK5-independent pathway to support cell survival and proliferation. However, the exact mechanisms need to further explore.  Taken together, our results suggest that PAK5 is overexpressed in human glioma tissues. Moreover, the inhibition of PAK5 by lentivirus-mediated RNAi suppressed glioma development by weakening the anti-apoptotic ability of cells and promoting cell cycle arrest. This study provides a novel therapeutic target for gliomas, although the molecular mechanisms of PAK5 in gliomas require further investigation.

Abbreviations

Figure 1 .
1PAK5 is highly expressed in glioma cells. (A) The ratio of PAK5 mRNA expression in freshly sampled gliomas and their adjacent normal tissues from 40 glioma patients. (B) Representative immunohistochemical staining of PAK5 expression in human glioma tissues (upper) and pericancerous tissues (lower). (C) Western blot assay of PAK5 expression in glioma cell lines.

Figure 2 .
2Lentivirus-mediated RNAi significantly inhibits PAK5 expression in human glioma cells. (A) The lentiviral transduction efficiency in U87 cells was estimated 3 days after infection at an MOI of 5. Light micrograph (upper); fluorescent micrograph (lower) (× 400). (B) Total RNA of U87 cells was extracted at 4 days after infection, and relative PAK5 mRNA expression was determined by qRT-PCR. GAPDH was used as an internal standard. The data represent the mean ± SEM of three independent experiments. *, P < 0.05, **, P < 0.01. (C-E) Western blot analysis of PAK5 protein expression levels of Scr-shRNA-infected and PAK5-shRNA-infected cells in U87 (C), SHG-44 (D) and CHG-5 (E) cells.

Figure 3 .
3Inhibition of PAK5 remarkably suppresses cell viability in glioma cells with high PAK5 expression. (A-C) Cell viability curves of U87 (A), SHG-44 (B) and CHG-5 (C) cells during the 5 days were evaluated by the MTT assay. The data represent the mean ± SEM of three independent experiments. *, P < 0.05 vs. cells infected with Scr-shRNA lentivirus. (D) PAK5 protein expression in U87 cells infected with a vector with expressing PAK5 after PAK5-shRNA1-mediated PAK5 knockdown. (E) Cell viability curves of U87 cells of three groups during the 5 days were evaluated by the MTT assay. The data represent the mean ± SEM of three independent experiments. *, P < 0.05 vs. cells infected with Scr-shRNA lentivirus.

Figure 4 .
4Inhibition of PAK5 prominently suppresses tumor development in U87 cells. (A) Colony formation assay of cells from the three groups. Cells were seeded at 500 cells/well and allowed to form colonies for 7 days. The colonies were stained with crystal violet and counted. Data represent the mean ±SEM of three independent experiments (right). **, P<0.01. (B) Cell cycle analysis of cells from the three groups as determined by PI staining and FACS analysis (left). The percentages of cell cycle phases represent the mean ± SEM of three independent experiments (right). *, P < 0.05. (C, D) Scr-shRNA-infected and PAK5-shRNA-infected groups of nude mice were injected with the corresponding infected U87 cells (10 6 cells per mice) into the axilla. After 3 weeks, some mice were sacrificed, and their tumors were examined (n=6) (C). The other mice were used to detected the time of visible tumors (diameter > 3 cm) (n=6) (D). **, P < 0.01.

Figure 5 .
5PAK5 silencing increases cleaved caspase 3 levels and decreases β-catenin levels in U87 cells. Cell lysates ofScr-shRNA-infected, PAK5-shRNA-infected and PAK5-shRNA1-PAK5 vector-co-infected U87 cells were immunoblotted with the indicated antibodies.

Table 1 .
1Associations between PAK5 expression and clinicopathological characteristics of 40 glioma patientsClinicopathological
parameters

n
down 0-1
1-2
>2
P value

age
<60
32
5
2
5
20
>0.05
>60
8
1
0
2
5
gender
male
26
3
1
4
18
>0.05
female
14
3
1
3
7
grade
I-II
16
3
1
3
9
>0.05
III-IV
24
3
1
4
16

P value was detected by chi-square test

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AcknowledgmentsThe authors thank Dawn S. for critical reading and editing of the manuscript.Competing InterestsThe authors have declared that no competing interest exists.
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New Insight into Microvascular Perfusion by Analyzing Pipe Flow in a Cylindrical Elastic Shell
September 14, 2018

Yanchun Hu
Anming Hu
Department of Orthopaedics
The Fifth People's Hospital
JinanChina

Shandong Experimental High School
JinanChina

Shenju Song
Department of Nursing
The Fifth People's Hospital
JinanChina

Yanxia Hu
Department of Infectious Diseases

Center for Disease Control and Prevention
JinanChina

Yanchun Hu
New Insight into Microvascular Perfusion by Analyzing Pipe Flow in a Cylindrical Elastic Shell
September 14, 201894F0DECEEA94763A7EE27DF98972508810.4236/jbise.2018.119020Received: August 9, 2018 Accepted: September 11, 2018MicrovascularPerfusionHypertensionCylindrical Elastic ShellFluid Flow
Background: Microvascular perfusion, a kind of regional perfusion, plays important roles in delivering oxygen and nutrients, and regulating blood pressure and responses to inflammation.Aim: The aim of this research is to analyze the characteristics of microvascular perfusion by conducting pipe flow in a circular elastic tube.Methods: A model was established with circular elastic tube to mimic microvascular perfusion.The velocity of pressure waves was calculated according to the time that the liquid took to spilt over.What's more, the characteristics and significance of microvascular flow and arteriovenous anastomoses (AVAs) were analyzed.Results: It took the liquid about 0.1 second to spill over from the model, and the velocity of pressure waves is greater than 100 m/s in the elastic pipe.A mechanical switch structure and the corresponding mechanism were proposed for microvascular perfusion in AVAs.Conclusion: Microvascular perfusion maintains a considerable constancy of hemodynamics in different tissues, when ventricular contraction changes perfusion pressure to meet metabolic demands appropriately.This theory will help us to gain a new perspective in microvascular flow.

INTRODUCTION

Cardiovascular function and tissue perfusion are strongly affected by microvascular blood flow resistance, which in microcirculation is governed by flow behavior of blood through a complex network of vessels [1].The main function of the microvascular is to transport materials.Water and solutes are carried by blood through the microvessels and exchanged, through vessel walls, with the surrounding tissues.This transporting function is highly dependent on the architecture of the microvasculature and on the biophysical behavior of blood flowing through it.The microvasculature of a perfused tissue provides a large area for exchange of materials, and brings blood into close proximity with each point in the tissue.

Pipe flow is a type of liquid flow within a closed conduit, and it belongs to hydraulics and fluid mechanics.Due to the existence of friction, pipe flow should be driven by a pressure difference.Pascal's law states that the pressure exerted anywhere in a confined incompressible fluid is transmitted equally in all directions throughout the fluid.When fluids are in motion, Pascal's principle does not necessarily apply.Pascal's law, applied in fluid mixtures, is a meanfield theory of amphiphilic action, and interface wetting in glassy liquids, which allows fluids to transmit force through the media like pipes or tubes.For instance, a force applied to a fluid in a pipe can be transmitted to the other end of the pipe via the fluid [2].

It is known that an elastic cylindrical lossless shell immersed in an acoustic fluid can guide acoustic energy longitudinally without attenuation.In fact, for the physical and geometric parameters within suitable ranges, different surface wave modes, with different angular dependence around the cylinder, can be supported by the structure.These modes propagate longitudinally with subsonic phase velocities, and are evanescent in the transverse direction.

For a rigid circular pipe, if it is relatively longer, the value added in the entire pipeline will need some time to build.The value of fluid pressure is the transfer of intermolecular force between molecules, the intermolecular forces gradually spread to the distance, to the end of the pipe.It needs some time for the stress to be fully established.The added value of the pressure in the rigid perfect tube can be regarded as the low frequency pressure wave.Its transmission speed is equal to the speed of sound [3].To understand the dynamics of blood is a crucial element to discover biological mechanisms, to fully understand hypertension, and to design sophisticated microfluidic devices, for diagnostics.

MATERIALS AND METHODS

For this research, materials were used as follows: elastic circular tube, rubber tube, connecting pipes, switch, cylindrical containers, a stopwatch, needle electrode, and invasive blood pressure monitor [4].Tubes with the same external diameter (6 mm) and inner diameter (4 mm) were used.The studied samples are made of rubber.Also, an invasive blood pressure monitor was placed in the A side of the tube.The schematic diagram of the equipment is shown in Figure 1.

The elastic circular tube was adopted to take the rubber tube (AD, internal diameter 4 mm × external diameter 6 mm, AD was 20.2 meters long).The rubber tube BC as shown in Figure 1 was horizontal placed, which was 20 meters long.AB was perpendicular to the ground, and was 0.1 m high equal to CD, which was also perpendicular to the ground.Thus a communicating vessel was formed.The end A was connected with a water container M, and the water height was 0.3 m.There was a switch at the entrance A, and it was shut.A, D were remained at the same level.So, if it was switched off, there would be 0.1 m high water pressure inside horizontal pipe.After it was switched on, the time that the fluid took to overflow from D was recorded.In order to facilitate the observation, elastic pipe to take back, so that both ends were near to each other.And all the tubes were fixed on a rigid plate tightly as shown in Figure 2. A invasive blood pressure monitor was placed in the A side of the tube.The rise of the A end pressure was the

RESULTS AND DISCUSSION

A straight rigid circular pipe filled with water and pistons were placed at each end, as a consequence, pressure exerted against the left piston would be transmitted throughout the liquid and against the right piston as in Figure 3.If the pipe was 1000 m long, the left piston began to move about 1 sec later after the right side of the applied stress.Notably, the pistons are simply "plugs" that can slide freely but snugly inside the tube.When the water is just beginning to flow, the pressure that the left piston exerts against the water will be exactly equal to the pressure the water exerts against the right piston.Or when the water has been flowing, the pressure that the left piston exerts against the water will be greater than the pressure the water exerts against the right piston.

Pressure wave, a type of longitudinal waves (longitudinal waves are waves that have the same direction of vibration as their direction of travel), is formed from alternating compressions and rarefactions.In rigid pipeline, the pressure wave will travel in the medium at a velocity C equal to the velocity of sound in an infinite expansion of the medium, and given by an equation (α):
C K ρ =
where K = Bulk modulus of liquid, ρ = Mass density of fluid [5] [6].If the pipe is elastic, the velocity of propagation will be less than that given by the equation α.The result we obtained showed that it took about 0.1 s from the opening of switch at A to starting to flow out water at end D.

As shown in the above experiments, because D is open to the air, the entire pipeline cannot form the same hydrostatic pressure as the A. When D end starts to flow out water, the stress field is not fully established in the whole pipeline.For elastic pipe, flow out water from D end means the pressure starts to rise at the end of pipe.This is sufficient to change the balance of the end pressure to make the water flow out of the water.The pressure transmission speed is quite fast.In a very short time to produce pressure difference in tube end that pushes the liquid to move forward.

In the human body, the arterial blood pressure pulse is one of the most fundamental physical quantities related to haemodynamics.Measurement techniques have been developed, which at times have obscured the true nature of this pulsatile phenomenon.Blood pressure is, however, a composite wave that undergoes significant changes in its contour while traveling within the arterial system.Thus, the term "blood pressure", when used alone, does not express this wave nature.Pulse waves, which are usually measured, are waves that are oscillating perpendicularly to the direction of propagation.The pulse waves caused from LV (left ventricular) ejection that increase the arterial wall linearly [7].Pulse wave velocity (PWV) is obviously lower than the speed of sound, for a health younger, pulse wave velocity is less than 20 m/s in arterial [8].

In order to detect pulsation changes in blood volume in the microvascular tissue bed, the photoplethysmography (PPG) is often used [9].Because of the ventricular contraction, a volume of blood is squeezed into the periphery.Then microvascular perfusion get to peak that can be recorded by PPG.Combined with simultaneous cardiac ultrasound, Mitral valve closure can be measured as a starting point for increased ventricular pressure.PPG peak can be taken as a sign of the peak value of microcirculation perfusion, it appears only 0.2 seconds after aortic valve opens.This makes tissue perfusion almost at the same time, not because of the distance from the heart is different.Meanwhile, peak of PPG was earlier than pulse wave get to the periphery.

The capillary net is a strongly bifurcate network of small vessels, and the number of vessels in the capillary system is significantly larger than in the arterial and venous system [10].As a consequence, the total cross section in the capillary system is about 1000 times larger than in the arterial and the venous system.When the blood flows out of the capillaries into the venules, the total cross-sectional area will gradually become smaller.In this way, the capillary network has formed the pipeline structure which has an enlarged total cross-sectional area in the midsection and lesser total cross-sectional area at each end.When the blood flows through the lumen of a sudden enlargement of the area of the cross section, the blood flow in the diffuser is generated.Due to constant flow through the expansion of the lumen, it will inevitably lead to a reduction in the blood flow velocity.Blood flow through the cross-sectional area with a sudden decrease, will produce a convergence of the flow cross-section, such as the cone shape pipeline blood flow velocity distribution.

Due to small elastic modulus and larger cross-sectional area, the capillaries can behave as a "windkessel", which can cushion the pulsations generated by the heart.When the ventricle contracts, the pipeline of capillary net expands, and the highest value of capillary pressure in capillary network was lower than in the arterioles.According to basic flow equation, it cannot be concluded that capillary network flow resistance is less than the arterioles.The basic flow equation (Q = ΔP/R, blood flow = pressure/resistance) summarizes the relationship between the pressure difference, blood flow, and vascular resistance.The basic flow equation is originated from Ohm's law, based on a fundamental law of physics, which states that the current (I) equals the voltage difference (ΔV) divided by the resistance (R).In electrical circuits, Ohm's law is applied in the field of mathematics.However, the water analogy can create misconceptions, and awareness that electricity and water behave very differently is important [10].

Loss of mechanical energy of unit mass fluid in the process of movement is called head loss.The kinetic energy of the blood flow generated by the contraction of the heart is ultimately converted into heat because of friction.Order of magnitude, red blood cells with diameters of 7 µm must deform to pass through them.Head loss will be large in capillary network [11].

Capillaries are composed of a single layer of endothelial cells which lack of vascular smooth muscle.There is no neural or local metabolic control of capillary vessels, whereas arterioles have much thicker walls with smooth muscles.If we believe the resistance is highest in the capillaries, then why relaxation of vascular smooth muscles should lower blood pressure must be answered.

Mostly, blood develops to become the arterioles in two ways.The first way is to follow a metarteriole forms a specific part of the tissue and through the capillary system.The second way is to enter an arteriovenous anastomosis (AVA) that shortcuts the arterioles and the venules to bypass the tissue [12].Arteri-ovenous anastomoses (AVAs) are low-resistance vessels.They have a relatively larger diameter than capillaries, and they form low-resistance channels by which the high-capacity venous bed can be rapidly perfused.The structure of the blood vessel wall has different elastic modulus.A sphincter-like muscle was formed in the arterioles.It can adjust blood flow into controlled arteriovenous anastomoses.Histologically, AVAs are characterized by an irregular lumina (3 -8 times larger than those of capillaries) and muscular walls.In cross-sections, the thick walls of the arteriovenous anastomoses consist of circular and longitudinally arranged smooth muscle fibers, the opening is usually narrow and has a stellate outline due to the ridges of smooth muscle cells that protrude into the lumen.There is a notable thickening of the vascular smooth muscle at the origin of AVAs from the parent arterioles.

The thickening tissue of the vascular smooth muscle in the parent arterioles makes it possible to form a lip-type structure.The parent artery, the vascular smooth muscle differs from the endothelial cells in the elastic modulus.The vascular wall tension of the parent artery varies with the intravascular pressure over the cardiac cycle.Therefore, it is structurally possible for the AVAs to close during systole (blood pressure ≥ 90 mmHg) and to open during diastole (blood pressure < 90 mmHg) due to higher and lower wall tension in the arterioles, respectively [13].

In fact, if the systolic blood pressure becomes reduced (<90 mmHg) due to bleeding during shock, the arteriovenous anastomoses will open.This phenomenon is characteristic of changes in the microcirculation during shock [14].Based on the previous studies we conducted, vascular endothelial dysfunction represents an initial step toward hypertension.The endothelium plays multiple pathological and physiological roles, including the regulation of smooth muscle tone.Vascular smooth muscle relaxation or contraction may result in wall tension in arterioles, which also change arteriovenous anastomoses [16].

A number of unresolved issues still remain, particularly with regard to the relationship between blood flow properties as observed in vitro and the functional behavior of blood in the living microcirculation.

Future studies should address these issues, including the causes of the discrepancy between the apparent viscosity of blood in microvessels in vivo and that in glass tubes.The in vivo significance of red cell aggregation and sedimentation in both normal and pathological situations remains to be debated.Although measurements of the bulk viscosity of blood and red cell deformability are frequently correlated with specific clinical conditions, the actual significance of such measures with regard to blood circulation is poorly understood.The mechanics of red cell screening in microvascular bifurcations is not well understood, although this phenomenon is probably very significant in smaller bifurcations.The high level of heterogeneity in microvascular hemodynamics is recognized, but efforts to understand its implications for the mass transport functions of the microcirculation are at a relatively early stage.

In this work, we focused on the pipe flow in shell to gain a new perspective in microvascular flow, and the results demonstrated that microvascular perfusion maintains a considerable constancy of hemodynamics in different tissues from head to toe after ventricular contraction.Although it is difficult to get a specific answer or answers to microvascular hemodynamics, understanding the dynamics of blood cells is still a crucial element to discover biological mechanisms.It was reasonable to believe that changes of arteriovenous anastomoses were the key to the development of hypertension.With the development of modern science, branches of medical science have been increasingly coming up with their theory based on accurate physics and pipe flow analysis will help us fully understand the micro circulation.

Figure 1 .
1
Figure 1.Schematic diagram of the equipment.

Figure 2 .
2
Figure 2. Geometry of the model.

Figure 3 .
3
Figure 3. Schematic diagram of pipe with pistons.

Figure 4
4
showed a tension switch model with different elastic modulus materials.The two valves are symmetrical, as the valve channel between cd and c'd'.a, a' is the fixed axis, triangular acd and a'c'd' are valves and could rotate around a, a' fixed axis.The lines bc and b'c' were rubber rope (mimic endothelial cells), bd and b'd' were cotton rope (mimic vascular smooth muscle).Cotton ropes were longer rubber ropes.The elastic modulus of cotton rope is larger than rubber rope.There is a certain elastic F which generates pressure < 90 mmHg.The length: ac = ad, bc = bd.The stress of cotton rope is also F. The cd and c'd' parallel channel in the open state.The b and b' points in the opposite direction were respectively applied with forces F and F', respectively, which could generate a ≥ 90-mmHg pressure intensity in each direction.Due to the elastic modulus of cotton rope was larger than that of rubber rope, under the action of the tension, the valve acd would show a clockwise rotation, while a'c'd' would show a counter-clockwise rotation, resulting in a closed channel.When F and F' decreased, the channel was opened up with the counter-clockwise rotation of acd and clockwise rotation of a'c'd' [15].

Figure 4 .
4
Figure 4.The tension switch model for arteriovenous anastomoses.

CONFLICT OF INTERESTWe all declare that we have no conflict of interest.
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Starting with ourselves in deepening our understanding of generativity in participatory educational research

Linda Van Laren firstname.lastname@example.com
School of Education
University of KwaZulu-Natal
South Africa

Ronicka Mudaly
School of Education
University of KwaZulu-Natal
South Africa

Kathleen Pithouse-Morgan
School of Education
University of KwaZulu-Natal
South Africa

Shakila Singh
School of Education
University of KwaZulu-Natal
South Africa

Starting with ourselves in deepening our understanding of generativity in participatory educational research
9BD79CD5AB368062943E1DB1FE0D5F20generativityHIV&AIDSparticipatory researchreflexivitystoryboardinguniversity educatorsvisual methods
Participatory educational research is generally characterised by a commitment to making a difference in the lives of those who participate in the research and more broadly, to promoting social transformation.This suggests a potentially fruitful synergy between participatory educational research and the multidisciplinary body of academic work on generativity as a human capacity that has at its core a desire to contribute to the well-being of others.As a research team of teacher educators from diverse disciplinary backgrounds, we seek to add an alternative dimension to current debates on participatory educational research by focusing on understanding the 'how' and 'what' of generativity in a participatory research process.The research question we address is: How does/can engagement in participatory educational research facilitate generativity?While participatory research literature often concentrates on collaboration between researchers and 'researched' communities, we are taking a reflexive stance by exploring our own participation in our dual roles as university community members and as researchers studying our colleagues' experiences in relation to integration of Human Immunodeficiency Virus (HIV) & Acquired Immunodeficiency Syndrome (AIDS)-related issues in university curricula.We describe how our use of the visual method of storyboarding facilitated insight into generativity in participatory educational research.Building on an earlier concept of generativity, we identify and discuss significant generative features of participation, playfulness, passion, and perspicacity in our research process.

Introduction

Researchers concerned with responding to educational challenges, faced by learners, teachers and other community members, often choose participatory research methodologies to involve these stakeholders in working together to find context-appropriate ways to address these issues (e.g.Mitchell, De Lange, Moletsane, Stuart & Buthelezi, 2005;Theron, 2012).Such participatory research tends to be characterised by a commitment to making a difference in the lives of those who take part in the research and, more broadly, to promoting social transformation (Bergold & Thomas, 2012).This emphasis on making a difference suggests a potentially productive synergy between participatory educational research and the multidisciplinary academic work that has been inspired by Erikson's (1963) concept of generativity as a human capacity that is rooted in "a calling to contribute to the well-being of others, particularly younger people" (Pithouse-Morgan & Van Laren, 2012:417).As a research team of teacher educators from different disciplinary backgrounds, we seek to add another dimension to existing discussions of participatory educational research by exploring the 'how' and 'what' of generativity in a participatory research process.

While literature on participatory research frequently focuses on "community members, or stakeholders in communities, [collaborating] with researchers in addressing needs and enhancing resilience and well-being in societies" (Ferreira, 2012:512), in this article we take a reflexive stance to examine our own participatory engagement as researchers and university educators in a process of creating storyboards.In so doing, we expand on what is typically conceptualised as participatory research.As highlighted by various authors, including Cooke and Kothari (2001), Walsh (2012) and Milne (2012), a key tension in participatory research is the researcher/researched divide, so that often the study of issues of knowledge production and even participation itself carry this burden.Here we explore what happens when a research team goes beyond the researcher/researched tensions by focusing on its own participatory engagement.

In this article we explore the following research question: "How does/can engagement in participatory educational research facilitate generativity?"We begin by explaining the context of our inquiry.We then clarify our understanding of storyboarding as a visual, participatory research method and go on to describe our participatory storyboarding process.Next, we present and explain the two storyboards that we created.To follow, we identify and discuss significant generative features of participation, playfulness, passion, and perspicacity in our storyboarding process and consider how storyboarding as a visual, participatory method facilitated generativity.Finally, we offer some concluding thoughts on the value of exploring visual, participatory methods in relation to the concept of generativity in research.

Putting our research into context

We are a team of five university teacher educators who work in a school of education at a South African university.While there are some interdisciplinary connections, we teach, research, and supervise postgraduate students in different disciplines within the field of education, namely, Gender Studies, Languages and Arts, Mathematics, Science, and Teacher Development Studies.We are, however, united in our commitment to integrating HIV&AIDS education across disciplines.In this article we focus on our experience of using storyboarding as a visual, participatory method to follow up our research into the experiences of our colleagues who integrate social aspects of HIV&AIDS into their teaching of various subjects.

In our view, addressing the resilience and well-being of students is a priority for university educators in South Africa, where in 2012 approximately 5.6 million people were reported as being infected with HIV (Averting HIV & AIDS (AVERT), 2012), and in 2010 the mean HIV prevalence rate for university students across the country was found to be 3.4% (Higher Education HIV/AIDS Programme, 2010).Hence we argue that it is crucial for all students to have a critical understanding of HIV&AIDS and related social issues when negotiating their lives and work in a context of high HIV prevalence.

Framed within a recognition of the role of university communities in addressing HIV&AIDS, we build on work completed for a larger project where we aim to document and encourage the integration of HIV&AIDS-related issues in university teaching.As university educators who are committed to addressing HIV&AIDS through our own teaching, our common research interest lies in learning more from colleagues who share these concerns.In a previous article focusing on the personal and professional lived experiences of seven colleagues, we highlight critical aspects of their stories of engaging in what we describe as curriculum innovating (Van Laren, Mitchell, Mudaly, Pithouse-Morgan & Singh, 2012).The emotional and professional challenges and benefits that our colleagues shared during interviews led us to conclude that there are risks and rewards involved in integrating HIV&AIDS-related issues in university curricula, and in particular, we drew attention to the psychosocial benefits and challenges for educators.These were discussed within three themes, namely: "selfing", "distancing", and "valuing" (Van Laren et al, 2012:138).Selfing referred to university educators' renewal or change in their professional and personal selves as they responded to issues related to HIV&AIDS among students.Distancing signalled many students' reluctance to engage with the topic of HIV&AIDS, which was reflected the way in which they separated or distanced themselves from the epidemic, thus creating a challenge for university educators who attempted to integrate HIV& AIDS in university curricula.Distancing also drew attention to how educators who integrated HIV&AIDS into their curricula often experienced isolation from colleagues who refused to do the same on the basis that this integration diluted 'pure' disciplines.Furthermore, distancing linked to how educators often felt afraid of the emotional impact of integrating HIV&AIDS and unsure of how they would manage the possible responses of students to this emotionally laden topic.Valuing emphasised how, despite perceived personal and professional risks, university educators in this study viewed the integration of HIV&AIDS as a worthwhile, valuable endeavour, and invested prodigious effort in designing and facilitating modules in a way that addressed the social challenges brought by HIV&AIDS.Hence, in the conclusion to the article we argued for the significance of these benefits and challenges being "sensitively, yet openly addressed in a university community" (Van Laren et al., 2012:157).As a follow-up to this article, we decided to respond to our own recommendation by trying out the participatory visual method of storyboarding to envisage possible ways of stimulating discussion and interaction among university educators and leaders in relation to integration of HIV&AIDS-related issues in university curricula.

Storyboarding as a visual, participatory research method

Storyboarding is typically regarded as a stage in the process of filmmaking and, more recently, as a component of participatory video as a visual research method (Mitchell, 2011;Mitchell & De Lange, 2011).It is the planning component where film or video makers typically map out visually a series of shots that will tell a story.

Storyboarding as a research method has received limited attention, and the storyboard is rarely treated as a visual text in itself but rather as more of a 'means to an end', with the final video being the main visual research text.However, in a recent study Labacher, Mitchell, De Lange, Moletsane and Geldenhuys (2012) deliberately used storyboarding as a participatory method with teacher educators to address HIV& AIDS and curriculum integration.In this research the production of storyboards was the focus.In treating the storyboards as visual texts, Labacher et al. (2012) provide a framework for analysis that highlights three significant components: (1) pinpointing the problem and containing the issues; (2) resolution -what should be done?; and (3) ownership and engagement.

Our participatory storyboarding process

Responding to knowledge generated through qualitative research requires imaginative, resourceful ways to generate new insights.It calls for creativity, which can be facilitated by innovative and participatory methods (Bergold & Thomas, 2012;Hunter, Lusardi, Zucker, Jacelon & Chandler, 2002).We decided to use storyboarding as a visual, participatory method both to engage in reflection (Lillyman & Bennett, 2012) and to engage further with the conclusions we had arrived at in our previous article.

We began our five-hour research workshop with a discussion, where a team member introduced storyboarding and provided examples of its use in participatory video making.We established a working definition of a storyboard: a structured and preplanned visual outline or skeleton that comprises a series of drawings, sketches or explanations representing each scene or camera shot required to produce a video.Drawing on Mitchell and De Lange's (2011) steps for video making, we developed five steps for our participatory storyboarding process, as outlined below.

Step 1: Developing the prompt.This was our first key participatory activity.Given that we were building on our previous article, we started by reading aloud the Conclusion and Implications section to stimulate discussion about our storyboarding prompt.We developed the prompt: 'Beyond risky behaviour: HIV&AIDS curriculum innovating'.

Step 2: Forming storyboard groups.Given the size of our research team (five members) we divided into two smaller groups to prepare two different storyboards in response to the prompt.

Step 3: Creating the storyboards.Each group worked independently for two hours.We followed a procedure mapped out by Mitchell, De Lange and Moletsane (2011) and Labacher et al. (2012).This started with each group brainstorming a list of possible storyboard concepts.Each group then selected a particular concept to develop.Group 1 focused on 'Academically generative workshops' and Group 2 on 'Building collegiality and collaboration'.Next we prepared the storyboards, with each group providing a short title as a well as the genre and format of the proposed video.

Step 4: Presenting and reflecting.Each group presented its storyboard to the team and we discussed our selected theme, commonalities (and differences) and considered the next steps in terms of producing the videos.The discussion was recorded and transcribed for analysis.Later each team member wrote and emailed a reflection on her participation in the workshop.

Step 5: Making meaning.In considering the next steps we also contemplated how we could better understand the 'how' and 'what' of generativity in our own participatory engagement in the process of creating storyboards.We agreed that we had experienced the process as a generative one that held "[possibilities] for growth and development toward increased levels of potential knowing" (Ball, 2012:289).We felt that we needed to follow up by examining the concept of generativity in more depth and from a range of scholarly perspectives (Kincheloe, 2001).Hence, after the workshop we drew on a range of work from the domains of psychology and education (Ball, 2009;2012;Epstein, 1993;1999;Epstein, Schmidt & Warfel, 2008;Gergen, 1978;Strong, 2010) to assist us in exploring how our use of the visual method of storyboarding could facilitate insight into generativity in participatory educational research.Our data consisted of the storyboards that we developed collaboratively, transcripts of our recorded discussions during the workshop and individual team member's emailed reflections on our storyboarding workshop.

Ethical considerations and trustworthiness.The discussion in this article is based on our own observations, interactions and reflections during and after our workshop.This means that by exploring our dual roles as researchers and as university community members we did not "hinder or hurt" (Samaras, 2011:183) any other participants.However, we did have to be mindful of behaving in an ethical manner towards one another as colleagues and co-researchers during the workshop and while co-authoring this article.This meant that we had to check at every stage of the process that we were continuing to build "a sense of trust, security, and mutual respect" (Conle, Louden & Mildon,1998:191) within our team.

To enhance trustworthiness, we are drawing on Mishler's (1990) concept of trustworthiness for inquiry-guided (as opposed to hypothesis-testing) research.Mishler (1990:423) advises that trustworthiness requires researchers who are "engaged in inquiry-guided and interpretive forms of research [to articulate and clarify] the features and methods of [their] studies, of showing how the work is done and what problems became accessible to study".Accordingly, in this article, we endeavour to give a clear and detailed account of our research and meaning-making process.

What our storyboards portrayed

Group 1 considered playful and entertaining video genres, such as stop-motion photography, sock puppets or animation.The storyboard (Figure 1) 'Take a risk: It's as easy as ABC!' aimed to acknowledge risks and discomforts experienced by university educators in integrating HIV&AIDS and to encourage innovation and collaboration.South African Journal of Education;2013;33(4) In HIV prevention campaigns in South Africa the acronym ABC represents 'Abstain, Be Faithful, Condomise', and has been used extensively.However, research (Piot, Bartos, Larson, Zewdie & Mane, 2008;Reddy, 2005) suggests that this simplistic, overused message is "inappropriate … to manage an effective dialogue about sex" (Reddy, 2005:18).In this storyboard ABC represents 'Act, Be bold, Collaborate', which Group 1 identified as key to integration of HIV&AIDS in university teaching.Collaboration with colleagues was emphasised as a means of support for taking action, being bold, and responding to our recommendation of "[establishing] a learning community or a support group for university lecturers who take on the somewhat risky integration of HIV&AIDS issues in disciplines" (Van Laren et al., 2012:156).

Group 2's storyboard (Figure 2) depicts a television interview scenario.The 'actors' in the interview are an innovator (university educator who integrates HIV& AIDS), a resistor (university educator who does not integrate) and a facilitator (interviewer).The discussion in this storyboard underscores challenges related to integrating and offers the '3 Cs' (courage, conviction and collaboration) to address these challenges.Complexities and possibilities posed by the three Cs were debated during the interview.

Drawing on our storyboarding to gain insight into generativity in participatory educational research

Generativity has been researched in psychology from varied and often seemingly contradictory vantage points, including developmental psychology (Erikson, 1963;Erikson & Erikson, 1981), behavioural psychology (Epstein, 1993;1999;Epstein et al., 2008), social psychology (Gergen, 1978), and critical psychology (Strong, 2010).Interesting insights emerge from bringing these scholars' voices into dialogue with one another and with work done in the areas of teacher education and educational research (Ball, 2009(Ball, , 2012)).

This "multiperspectival" (Kincheloe, 2001:682) body of work on generativitywhich builds on the foundation of Erikson's conception of generativity (1963;Erikson & Erikson, 1981) -portrays it as a multifaceted human capacity, with three significant dimensions:

• responsibility -responsiveness to the needs of other people, particularly younger people; • productivity -contributing to the vocational/professional domain; and • creativity -envisaging and enacting new possibilities.Hence generativity can be contrasted with 'less desirable' human qualities such as selfcentredness, unproductiveness, and inertness.Erikson's (1963) work focuses on understanding generativity within his broader theory of psychosocial development.Drawing on the work of other scholars (Ball, 2009;2012;Epstein, 1993;1999;Epstein et al., 2008;Gergen, 1978;Strong, 2010), and our experience of storyboarding, we have identified four features of generative processes that point to possibilities for recognising and nurturing generative thinking

Participation

In our consideration of participation, we draw on the work of Ball (2009), Epstein (1999) and Strong (2010), who emphasise teamwork and collaboration in generative processes.Epstein (1999) argues that it is useful to alternate between working individually and working with others.He advocates frequent changes in social environment by working with different groups of people or different combinations within groups.Ball (2009:53) advises that we can learn from others by "[tapping] into the rich cultural and linguistic resources" they offer.Strong (2010) cautions that genuine participation requires us to explore alternative ways of interacting with others.

As an established research team we are acutely aware of the value of learning from others.Coming from different disciplinary backgrounds and having a range of research interests, we serve as valuable resources for one another.This concurs with Epstein's (1999) suggestion for promoting generativity by working with diverse groups of people.Likewise, Pithouse, Mitchell and Moletsane (2009:31-32) draw attention to how co-researchers from different backgrounds and locations can "[prompt] each other to engage in processes of cultural, linguistic, and disciplinary 'translation' and explication", which can "be a trigger for intellectual, professional, and personal inquiry, growth, and renewal".The following reflections show that we found value in working together:

Claudia: Taken as a whole, however, I really liked the session and I loved the way we worked together as a team in doing this.Ronicka: I did not feel threatened to step out of my familiar research terrain because I was working collaboratively with a group of friends, in a safe place.I benefitted from the kinship, enthusiasm and support offered by the entire group.

Playfulness

Playfulness, as an attitude, an atmosphere and as a way of doing things, makes a vital contribution to generativity.Strong (2010) and Epstein (1993;1999) emphasise the generative value of engaging regularly in imaginative or open-ended activities, without predetermined limits or outcomes.Epstein (1993;1999) also draws attention to the role of the physical environment in stimulating this kind of open-ended play.Playfulness can also be stimulated by learning new knowledge and ways of thinking or doing -outside our delineated 'areas of expertise'.This is what Epstein (1993:44) terms "broadening" and Strong (2010) explains as moving beyond our habitual thinking.Epstein (1993:44) argues that "the newer the knowledge -the more it differs from what we already know -the more interesting the [possibilities]" for imaginative play.

Particularly significant when engaging in participatory research is that because generative play involves risk-taking, it is essential to cultivate an atmosphere of trust and mutual respect (Ball, 2009;Epstein, 1993;1999;Strong, 2010).Participants must be able to try out new and different ways of doing things and offer fresh ideas without fear of ridicule or premature judgement.

We see playfulness as an appropriate way to describe our approach to storyboarding.As a team we explored imaginative ways to move beyond the challenges associated with integrating HIV&AIDS-related issues.We had to venture into our creative selves to become immersed in the process of storyboarding.We were able to move outside our familiar research boundaries because we worked collaboratively in a non-threatening environment.The following reflections reveal this playful element:

Kathleen: I recognised that storyboarding could offer a fun and interactive way of planning.Linda: I had fun working on our storyboard.We recognised that storyboarding also involved risk-taking.The following reflection shows a team member's enthusiasm about making use of an unfamiliar methodology, combined with feelings of riskiness because of her uncertainty about what to expect and how to proceed in using storyboarding:

Shakila: I approached the workshop activity with as much enthusiasm as uncertainty.The major contributor to the reduction of my uncertainty … was the collaboration with members of my group.The following reflections reveal that the creation of an enabling atmosphere, in which we participated collaboratively in an environment of mutual respect and trust, was essential to our generative research endeavour:

Claudia: I loved the way we worked together as a team.Shakila: … the increase in my enthusiasm was undoubtedly [due to] the collaboration with the members of my group.As a team we were therefore better able to cope with the risks in using an unfamiliar research method.Moreover, through working collaboratively in an area of research where we are passionate, we enjoyed the participatory process of storyboarding.

Passion

Playfulness has connotations of enthusiasm and enjoyment.To play a game well, one must be committed to and immersed in the game.Thus, Ball (2009;2012), Gergen (1978) and Strong (2010) draw attention to the central role of passion in generativity.Ball (2009:67) explains that generative thinking and action must be "grounded in work that [is] important… [and] of real interest" to those involved.Strong (2010) proposes that rigour in relation to generativity can thus be understood as acknowledging the social nature of the development of our interest in certain topics.

Each member of our team has a research interest in the area of HIV&AIDS and curriculum, and has published in this field.We share a genuine concern about integrating HIV&AIDS-related issues into university curricula in substantive and innovative ways, as opposed to ways that are purely symbolic or tokenistic.The following reflections provide examples of how we expressed our passion for sustaining and furthering HIV&AIDS integration: Kathleen: In an area such as HIV&AIDS, where there is often a sense of hopelessness, it is good to feel as if we can move forward, together.Linda: The morning workshop was purposeful and not just a 'talk-shop'.We had concrete actions for taking-up possibilities after the workshop and I believe that our storyboard team activity provided interesting and fun ideas for moving towards generativity.We were committed to becoming immersed in the participatory research process, and we did so with a great deal of passion, enthusiasm and delight, which is evident in our reflections.The following reflection reveals our eagerness about using the storyboards to develop videos:

Claudia: The other thing that interested me was the enthusiasm we all had for actually making the videos we had envisioned in the storyboarding process.

Perspicacity

In highlighting the significance of passion in generativity, Ball (2009;2012), Gergen (1978) and Strong (2010) are careful to also emphasise the need for cultivating perspicacity -or clear-sightedness and insightfulness -about what it is we cherish and the foundations and possible consequences of our passion.Gergen (1978Gergen ( :1356) ) contends that we must "confront more directly and honestly the valuational implications of [our] work" and "give self-conscious consideration to matters of value".Ball (2012:287) proposes that researchers should engage in a self-reflexive process of "reflection", "introspection", "critique" and developing "personal voice" to move beyond advocating change to becoming generative agents of change.Thus, even though playfulness and passion might suggest that fostering generative thinking and action is an uncritical process, perspicacity reminds us that heightened self-awareness and constructive critique from self and others are essential.Ball (2009) and Epstein (1993) draw attention to the importance of developing strategies for enhancing perspicacity.Epstein (1993:44) highlights the significance of finding simple and practicable ways to "capture" new ideas as they occur to us.He argues that all our new ideas, even if they do not seem very 'good' at the time, should be noted and can be revisited and evaluated at a later stage.He suggests notebooks, audio recorders and computer software as possible capturing tools.Ball (2009:66) illustrates how writing, in particular, personal narrative writing, can also be used as a "tool for reflection, introspection, and critique".

Our commitment to generating new possibilities for stimulating discussion and interaction among university educators in relation to integration of HIV&AIDS-related issues in university curricula necessitated the development of perspicacity (clearsightedness and insightfulness).Our storyboards show that we wanted to cultivate a deeper awareness of how we as university educators can work with colleagues to respond to challenges associated with HIV&AIDS integration into disciplines.

Our storyboarding was driven by the desire to turn our passion into creation of something valuable.There was deep commitment to progress beyond the interpretation of research findings and to use the experiences of HIV&AIDS integrators to become generative agents of change (Ball, 2012).For example, in our reflections, team members wrote:

Claudia: What struck me most about the activity was its relevance as a follow up tool to fieldwork/research… .So now I think we could pitch storyboarding as a way to go beyond data.

Linda: If we don't take-up or do something about the emotional stress experienced by our colleagues who are integrators of HIV&AIDS education then our research project work is pointless, because no one else will be willing or able to do something about the challenges of integrating HIV&AIDS education in disciplines.

Kathleen: I had a sense of purpose and saw storyboarding as something that would build on and contribute to our research -rather than as 'just an exercise'.Their reflections point to our desire to seek direct ways to encourage dialogue and interaction on the subject of integration of HIV&AIDS-related issues in university curricula.

Furthermore, our reflections reveal that throughout the workshop team members were aware of personal inadequacies, understandings and practices and how these might affect the storyboarding process:

Ronicka: I had to engage with skills such as drawing and scriptwriting, which were new to me.Shakila: However, as we began engaging with the task, I discovered that previous experience was not critical and that the activity took on a life of its own.These reflections show that we openly acknowledged our limited experience in storyboarding, but saw the workshop experience as an opportunity to develop and grow.Moreover, possible inadequacies and uncertainties did not deter us from learning through active participation in the storyboarding process.

How did storyboarding as a visual, participatory method facilitate generativity?

During our storyboarding workshop the team effort facilitated generativity in a variety of ways.First, we had previously gained knowledge about benefits, risks and discomforts experienced by our university educator colleagues who engage in curriculum innovating while integrating HIV&AIDS in curricula (Van Laren et al., 2012).Through storyboarding, we were able to envisage specific, pragmatic ways to provoke discussion of these benefits, risks and discomforts within and beyond our university community (Labacher et al., 2012).

Second, the process of creating storyboards allowed us to consider varied ways of stimulating dialogue and interaction in relation to integration of HIV&AIDS education (Labacher et al., 2012).Working as a team of researchers with different levels of expertise and disciplinary backgrounds contributed to a broadening of the epistemological and methodological research base of the team.The generation of two quite different storyboards could be attributed to the diversity of interests of team members.

Third, by 'starting with ourselves', we found that we could begin to take up the recommendations that we presented in our previous article (Van Laren et al., 2012) by, for example, establishing a learning community among ourselves as university educators who take on the integration of HIV&AIDS-related issues in our disciplines.We simultaneously 'moved forward' by generating storyboards that will be turned into videos to share ideas and provoke discussion with our colleagues and with university educators in other contexts.Moreover, while as noted above not all team members were familiar with storyboarding, we were successful in taking this work up as part of a participatory process.

Concluding thoughts

We have presented an exploration of storyboarding as a visual, participatory method that facilitated generative thinking and action within a research team of university educators.We have framed our findings within four features of generative processes, namely participation, playfulness, passion, and perspicacity.In a field of research such as HIV&AIDS-related education, within a context of the continuing high rate of HIV infection and the challenges that this presents to resilience and well-being in our society, it is not just useful but also necessary to move beyond familiar ways of thinking and working.As research has highlighted (Higher Education HIV/AIDS Programme (HEAIDS), 2010), while integrating HIV&AIDS into university curricula receives endorsement 'from the top' at the level of policy (Higher Education HIV/ AIDS Programme (HEAIDS), 2012), it can often be lacking in terms of practical support for educators and recognition of what it takes to transform one's classroom from one focusing primarily on a disciplinary area into one that is also meant to offer students strategies for dealing with social and personal challenges.We attempted to address this work 'head-on' by creating storyboards that acknowledge and suggest ways to stimulate dialogue and interaction in relation to the psychosocial demands of integration.

This article highlights and opens up new ways of thinking about the value of visual, participatory methods in relation to the notion of generativity in research by minimising some of the inherent constraints in researcher/researched contexts that may overshadow or confound the study of generativity.Ultimately of course, power and ethics are key areas of concern in participatory research.We have simply argued for ways of nuancing this work, and especially for supporting research teams to take a 'starting with ourselves' approach that can also contribute to theorising.This does not mean that we wish to ignore power differentials that often exist in participatory research; rather, we wanted to deepen an understanding of the 'how' and 'what' of participation in the absence of some of the constraints.Methodologically our inquiry confirms findings of previous work on the value of storyboarding in helping to identify solutions (Labacher et al., 2012), and it also has offered an analysis of the 'doing' itself.Thus it contributes to an emerging body of work on storyboarding as a visual, participatory method that both sits alongside and incorporates work on photovoice, digital storytelling, participatory video and drawing (Mitchell, 2008;2011;Theron, 2012).

Our small-scale 'starting with ourselves' storyboarding project also offers an example of the kind of reflexivity that we see is required for 'taking action' in contexts where rates of HIV infection are still unacceptably high and where it complements other work linked to reflexivity through participatory video (Yang, 2012;Milne, 2012;Mitchell & De Lange, 2011).At the same time it has helped us to envisage ways of giving a public face to this work (in the form of the final videos), which can be made available for screening and discussion in professional development sessions with higher education colleagues or online to reach a broader audience.

Figure 1
1

Figure 2
2
Figure 2 Group 2's storyboard: "Reducing risk through courage, conviction and collaboration

AcknowledgmentsWe acknowledge the contribution of our team member, Claudia Mitchell, who introduced us to storyboarding as a visual participatory method and who inspires us to continue learning and growing as researchers and educators.
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