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Cardiovascular Research 2007 73(3):481-487; doi:10.1016/j.cardiores.2006.10.011
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Copyright © 2006, European Society of Cardiology

Myocardial angiogenesis after plasmid or adenoviral VEGF-A165 gene transfer in rat myocardial infarction model

Xiaojin Haoa, Agneta Månsson-Broberga, Karl-Henrik Grinnemob, Anwar J. Siddiquia, Göran Dellgrenb, Lars Å. Brodinc and Christer Sylvéna,*

aDepartment of Cardiology, Karolinska University Hospital, M52, S-141 86, Stockholm, Sweden
bDepartment of Thoracic Surgery, Karolinska University Hospital, Stockholm, Sweden
cDepartment of Clinical Physiology, Karolinska University Hospital, Stockholm, Sweden

* Corresponding author. Tel.: +468 5858 0000; fax: +468 5858 6710. Email address: Christer.Sylven{at}ki.se

Received 4 May 2006; revised 28 September 2006; accepted 16 October 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: To compare gene transfer of plasmid (P) and adenovirus (Ad) encoding human vascular endothelial growth factor-A165 (hVEGF-A165) angiogenic efficacy and adverse effects as regards apoptosis and ectopic expression of the transgene in a rat myocardial infarction model.

Methods: Myocardial infarction was provoked in Fisher rats. One week later, PhVEGF-A165, PLacZ, AdhVEGF-A165, or AdLacZ was transferred intramyocardially along the border of the myocardial infarction. hVEGF-A expression was detected with ELISA. Myocardial vessel density was analyzed 1 and 4 weeks after gene transfer. Apoptosis was detected by TUNEL staining. Cardiac function was assessed with Tissue Doppler Velocity Imaging.

Results: Although AdhVEGF-A165 had substantially higher myocardial hVEGF-A expression than PhVEGF-A165, AdhVEGF-A165 and PhVEGF-A165 induced angiogenic effects to a similar extent with maintained increased arteriolar density after 4 weeks of gene transfer (p<0.05). The two treatments also improved left ventricular function similarly. Adenoviral gene transfer induced a higher number of TUNEL positive cells than plasmid (p<0.02). Ectopic expression of the transgene was present with both vectors but substantially higher after adenoviral gene transfer.

Conclusions: AdhVEGF-A165 has no obvious angiogenic advantage over PhVEGF-A165 but more side effects at least in a rat myocardial infarction model. This indicates that PhVEGF-A165 might be more applicable for therapeutic angiogenesis than AdhVEGF-A165.

KEYWORDS Angiogenesis; Gene therapy; Infarction; Growth factors; Apoptosis

This article is referred to in the Editorial by S. Nikol (pages 443–445) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Transient myocardial overexpression of human vascular endothelial growth factors A (hVEGF-A) by adenovirus (Ad) or plasmid (P) based gene transfer induces angiogenesis both experimentally and in patients with myocardial ischemia [1]. Double-blind randomized and placebo-controlled phase II clinical trials suggest that therapeutic angiogenesis takes place although these studies did not conclusively demonstrate clinical benefits [2–4]. Both types of vectors have been used in clinical trials for therapeutic angiogenesis. However, their angiogenic efficacy and side effects have not been directly compared in a myocardial infarction model. Adenoviral gene transfer can induce more effective expression of the transgene than plasmid [5,6]. But it is unclear whether higher intramyocardial hVEGF-A expression level following adenoviral gene transfer also may lead to more pronounced angiogenesis, although continuous retroviral myocardial upregulation of VEGF-A expression was reported to lead to hemangioma formation in immunodeficient mice [7]. Besides, little attention has been paid to potential vector side effects. Adenovirus can modulate apoptosis [8,9]. When adenovirus is used as gene transfer vector, its structure has been modified [10,11]. But whether the modified adenovirus or plasmid induces apoptosis that may be harmful to the myocardium remains to be investigated. In addition, differences in the degree of ectopic expression, another safety issue, have not been explored. This study compares the effects of plasmid and adenoviral gene transfer on hVEGF-A expression, angiogenesis, left ventricular function and vector side effects as regards apoptosis and the degree of ectopic transgene expression.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Adenoviral and plasmid vectors
Human GMP-grade first-generation adenovirus [12] or naked plasmids were used. Both vector types encoded LacZ marker gene or human VEGF-A165 [13], driven by a cytomegalo-virus (CMV) immediate early promoter.

2.2. In vivo gene transfer
All procedures involving the use and care of animals conform with Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by Stockholm Southern Ethics Review Board.

Male Fisher rats (200–250 g) were anaesthetized with midazolam (5 mg/kg) and medetomidine hydrochloride (0.1 mg/kg), and they were intubated and ventilated. The chest was surgically opened through the fourth intercostal space to expose the heart.

2.2.1. Experiment in non-infarcted heart
In order to determine the optimal doses of adenovirus or plasmid, 1x109, 5x109, 1x1010 pfu AdhVEGF-A165 or 20, 40, 60 µg PhVEGF-A165 in 100 µl saline was injected intramyocardially into the anterior wall at one spot in the normal heart. There were 5–6 animals in each group. Hearts were harvested to detect hVEGF-A expression 24 h after PhVEGF-A165 and 6 days after AdhVEGF-A165 gene transfer. These time points were previously reported to give maximum myocardial expression of the respective vector [14,15].

2.2.2. Experiment in infarcted heart
Left anterior descending coronary artery (LAD) was identified and ligated. Pallor and regional wall motion abnormality of the left ventricle confirmed occlusion. Intramyocardial gene transfer was designed to be delivered 1 week after myocardial infarction via a new thoracotomy. Earlier rethoracotomy before 7 days was not permitted by the Ethics Committee and direct delivery after myocardial infarction is not applicable in clinical situation. Treatment at a later time point with progressive cardiac remodeling and cardiomyocyte loss would have less therapeutic effects. Accordingly 7 days was chosen for gene delivery.

Rats were randomly selected for the four treatments. Transferred genes were: AdhVEGF-A165 (n=6), AdLacZ (n=6), PhVEGF-A165 (n=6), and PLacZ (n=7). Rats received 5x109 pfu adenovirus or 40 µg plasmid in 100 µl saline. A suture was made in the middle area of the myocardial infarction to make a landmark for the injection. Adenovirus or plasmid was injected intramyocardially with an insulin syringe (0.3 mm in diameter) in one spot into the peri-infarction region along the border of the myocardial infarction at the level of the suture (Fig. 1). Aspiration without retrograde blood filling of the syringe was used as a sign that the needle was in the myocardium but not in the left ventricular cavity. After 1 or 4 weeks animals were euthanized and hearts were collected. Hearts for histological analysis were frozen on OTC compound. Frozen sections were prepared.


Figure 1
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  		Fig. 1 Example of infarcted heart with peri-infarct region where injection was given (arrow). Haemtoxylin-eosin staining. 1.25x.

 
2.3. Human VEGF-A protein expression
Human VEGF-A gene expression was determined by ELISA in hearts both with and without myocardial infarction. In addition, ectopic hVEGF-A expression was determined in kidney, lung, liver and spleen from the non-infarcted rats. Organs were minced with a homogenizator knife in homogenization buffer. Thereafter the homogenized substance was centrifuged for 10 min at 14,000 xg rpm at 4 °C. The supernatant was collected and frozen at –70 °C. Later all frozen samples were analyzed by ELISA for hVEGF-A by immunoassay according to the manufacturer's instructions (Quantkine, R&D system).

hVEGF expression was visualized in myocardial tissue by immunohistochemistry stained with hVEGF antibody. Frozen sections were incubated with monoclonal anti-hVEGF-A (Sigma) followed by adding fluorescent-labeled (FITC) secondary antibody.

2.4. Endogenous VEGF-A protein expression
Endogenous VEGF was analyzed by ELISA with detection of rat VEGF-A (Quantkine, R&D system). The heart samples were obtained as for human VEGF-A.

2.5. Capillary and arteriolar densities
Animals with myocardial infarction were euthanized 1 or 4 weeks after gene transfer. For the analysis of capillary density the sections were incubated with Griffonia Badeiraea Simplicifolia Isolectin B4 (GSL-I-B4, Vector Laboratories) followed by a second incubation with ABC Complex. Finally, capillaries were visualized by DAB with supplementation of 0.03% hydrogen peroxide. Capillaries were counted at a magnification of 400x taken by an LCD camera (Olympus, Japan) connected with a microscope. Ten fields of pictures around the injection site were taken and the capillary count was analyzed blinded with an image analysis program (Micro Image, Olympus). For the analysis of arteriolar density, lectin stained sections were incubated with primary antibody against {alpha}-actin (Sigma). Then rabbit anti-mouse secondary antibody (FITC, Dako) was used to visualize the blood vessels. Blood vessels stained around the injection site were counted under 200x magnification in a fluorescence microscope. All analyses were performed in a blinded manner.

2.6. Apoptosis assay
TUNEL assay was performed on frozen sections with an ApopTag Fluorescein in Situ Apoptosis Detection Kit (CHEMICON International, CA, USA) according to the manufacturer's specifications. Sections of heart tissue were treated with digoxigenin-dNTP and termial deoxynucleotidyl transferase followed by incubation with anti-digoxigenin conjugated with fluorescein. Counterstaining with DAPI was performed to visualize nuclei. Five fields of apoptotic positive cells were counted under 400x magnification in a blinded fashion. Two areas along the border of the myocardial infarction were analyzed: the injection site and the contralateral site of the myocardial infarction where no injection had been given.

2.7. Characterization of apoptotic cells
After TUNEL staining, slides were incubated with anti-{alpha}-sarcomeric actin to detect if the apoptotic cells were cardiomyocytes. Then fluorescent-labeled (TRITC) secondary antibody was added for visualization of cardiomyocytes.

2.8. Left ventricular function
Cardiac function was assessed by Tissue Doppler Echocardiography using a Vingmed Vivid 5 (Vingmed A/S, Norway) ultrasound system equipped with a 10 MHz transducer. Echocardiography was performed on the rats 3 days after ligation, and 4 weeks after gene transfer. Color Tissue Velocity Image was performed at the apical chamber view at frame rates close to 230 fps. The probe position was adjusted under the guidance of Pulsed Tissue Doppler to the maximum velocity of the mitral valve plane. The recording was obtained by positioning a sample volume in each basal septal left ventricular wall segment. Analysis was performed off-line from acquired cine loops with Echopac version 6.1. Peak systolic velocity was not measured due to unfavorable noise to signal ratio. Systolic velocity–time integral, a parameter for displacement of myocardium which is less noise dependent, was assessed from 2–3 beats to minimize the variability. The final value of regional systolic myocardial function was calculated as a mean value from 2–3 peak velocity–time integral at systole.

2.9. Statistical analysis
Data are presented as mean±SEM. Comparison between the groups was made by means of Student's t-test, 1-way ANOVA followed by Fisher's PLSD test. Paired t-test was performed to compare the difference between values before and after treatment. Values were considered to be significantly different at a value of p≤0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. hVEGF-A expression
Fig. 2 shows AdhVEGF-A165 and PhVEGF-A165 dose–response curves for VEGF-A expression. The highest adenoviral dose, 1x1010 pfu, was associated with respiratory symptoms; the lowest dose, 1x109 pfu, was considered to have suboptimal hVEGF-A expression. The middle dose, 5x109 pfu, was then chosen as the optimal dose for further study. Of the plasmid doses the middle dose (40 µg), was chosen for its numerically highest hVEGF-A expression (p<0.01).


Figure 2
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  		Fig. 2 hVEGF-A expression in hearts after treatment with different doses of PhVEGF-A165 (20, 40 and 60 µg) and AdhVEGF-A165 (109, 5x109, and 1010 pfu). Values are mean±SEM.

 
AdhVEGF-A165 induced substantially higher hVEGF-A expression than PhVEGF-A165 in normal myocardium (17,280±5467 versus 890±452 pg/ml, p<0.001, Fig. 3A). In peri-infarction myocardium, AdhVEGF-A165 and PhVEGF-A165 expression was reduced to about 7% from that in the normal myocardium (938±290 versus 60±13 pg/ml, p<0.01). Still AdhVEGF-A165 had a higher expression than PhVEGF-A165. hVEGF-A expression can also be visualized in myocardial tissue by immunohistochemistry (Fig. 3B).


Figure 3
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  		Fig. 3 hVEGF-A expression in hearts. (A) hVEGF-A detection with ELISA in hearts without and with myocardial infarction after gene transfer of PhVEGF-A165 (24 h) or AdhVEGF-A165 (6 days). Values are mean±SEM. **p<0.01, ***p<0.001, AdhVEGF-A165 versus PhVEGF-A165. (B) hVEGF expression was visualized in myocardial tissue by immunohistochemistry stained with anti-hVEGF after gene transfer of AdhVEGF-A165.

 
Following intramyocardial delivery into the normal myocardium, ectopic expression of hVEGF-A in the kidney, lung, liver, and spleen was in the range of 2–5% of the myocardial expression. However, AdhVEGF-A165 still induced higher hVEGF-A expression than PhVEGF-A165 (318–503 for AdhVEGF-A165, 25–58 pg/ml for PhVEGF-A165, p<0.01, Fig. 4).


Figure 4
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  		Fig. 4 Ectopic expression of hVEGF-A in non-target organs after intramyocardial gene transfer of PhVEGF-A165 and AdhVEGF-A165. Values are mean±SEM. **p<0.01, ***p<0.001, AdhVEGF-A165 versus PhVEGF-A165, Student's t-test.

 
3.2. Endogenous VEGF-A protein expression
For whole heart endogenous VEGF expression in normal myocardium, post myocardial infarction without or with injection of pLacZ or adLacZ did not differ (323±18, 294±10, 313±70 and 272±19 pg/ml, respectively). AdhVEGF-A165 induced a higher level of rat VEGF-A expression (453±47 pg/ml, p<0.02) with a net increase of about 150 pg/ml being about 20% of the hVEGF-A expression. A similar pattern was seen for phVEGF-A165 (348±22 pg/ml), although the increment was not significant which might at least partly be due to dilution of endogenous rat VEGF from non-infarcted parts of the myocardium.

3.3. Arteriolar densities
One week after gene transfer, both PhVEGF-A165 and AdhVEGF-A165 induced about 80% higher arteriolar density compared with PLacZ (Fig. 5A, 28±2 for PhVEGF-A165, 25±4 for AdhVEGF-A165 versus 15±4 arterioles/mm2 for PLacZ, p<0.05). One month after delivery, arteriolar densities were still at higher level after both PhVEGF-A165 and AdhVEGF-A165 treatment compared with PLacZ or AdLacZ (p<0.05). AdLacZ showed a transient increase in arteriolar density at 1 week but not at 4 weeks.


Figure 5
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  		Fig. 5 Arteriolar (A) and capillary densities (B) 1 and 4 weeks after gene transfer of PhVEGF-A165, PLacZ, AdhVEGF-A165, and AdLacz. Values are mean±SEM. *p<0.05, 1 way ANOVA followed by Fisher's PLSD test.

 
3.4. Capillary densities
One week after gene transfer, both PhVEGF-A165 and AdhVEGF-A165 induced 17–18% higher capillary density compared with PLacZ (Fig. 5B, 2092±104 for PhVEGF-A165, 2117±91 for AdhVEGF-A165 versus 1784±41 capillaries/mm2 for PLacZ, p<0.05). After 1 month of treatment, there were no significant differences between any of the hVEGF-A165 and the LacZ treated groups. AdLacZ showed a transient increase in capillary density at 1 week but not at 4 weeks. No hemangioma was observed in any of the treatment groups.

3.5. Apoptosis
Apoptotic cell numbers did not differ between groups in the non-injected peri-infarction region (5–7 cells/field). Plasmid and saline injection did not significantly increase the apoptotic cell number compared with the non-injected site, but both AdLacZ and AdhVEGF-A165 induced a higher number of apoptotic cells (31±6 and 24±8 cells/fields, respectively; p<0.001, Fig. 6). Both AdLacZ and AdhVEGF-A165 effected higher apoptotic cell numbers than PLacZ or PhVEGF-A165 (p<0.02). No significant difference between AdLacZ and AdhVEGF-A165 was found.


Figure 6
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  		Fig. 6 Apoptotic cells in the border zone of the myocardial infarction 1 week after gene transfer in injected and contralateral non-injected area. TUNEL positive cells after treatment by AdLacZ in myocardial infarction border zone in (A) injected and (B) non-injected area, 400x. (C) Counted positive cells in non-injected and injected area after treatment with saline, PLacZ, PhVEGF-A165, AdLacZ and AdhVEGF-A165. Values are mean±SEM. ***p<0.001, versus non-injected area, {dagger} p<0.05, {ddagger}p<0.01 versus saline.

 
3.6. Characterization of apoptotic cells
Immunofluorescence double staining showed some apoptotic cells were positive for anti-{alpha}-sarcomeric-actin, which is specific for {alpha}-cardiac muscle actin but does not cross react with smooth muscle cells (Fig. 7).


Figure 7
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  		Fig. 7 Double staining for TUNEL (green) and anti-{alpha}-sarcomeric actin (red). Nuclei are DAPI stained (blue). Double stained cells are marked with arrow.

 
3.7. Left ventricular function
Left ventricular velocity–time integral deteriorated with time around 30% with PLacZ treatments compared with baseline (p<0.05, Fig. 8), whereas it improved with AdhVEGF-A165 (p<0.05) or PhVEGF-A165 (p<0.05) to around 50–60%. It did not change with AdLacZ. Four weeks after treatment, ventricular velocity–time integral increased both after PhVEGF-A165 and AdhVEGF-A165 treatment compared with PlacZ (p<0.05).


Figure 8
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  		Fig. 8 Fractional change of systolic velocity–time integral 4 weeks after gene transfer of PhVEGF-A165, PlacZ, AdhVEGF-A165, and AdLacz. *p<0.05, versus PLacZ, ANOVA followed by Fisher's PLSD test.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Intramyocardial AdhVEGF-A165 gene transfer induced substantially higher hVEGF-A protein expression than PhVEGF-A165 both in the myocardium and systemically. Unexpectedly, AdhVEGF-A165 and PhVEGF-A165 stimulated myocardial angiogenesis and improved cardiac function to a similar extent. AdhVEGF-A165 induced significantly higher level of apoptosis than PhVEGF-A165.

This experiment compares angiogenic efficacy at the doses of plasmid and adenoviral vector expressing optimal amounts of hVEGF-A. Based on previous experience, maximum expression following intramyocardial application in animal models occurs between 24–48 h for plasmid and 6 days for adenoviral vector [14,15]. AdhVEGF-A165 induced a dose-dependent increase in myocardial hVEGF-A expression. With the highest dose (1x1010 pfu), however, signs of systemic infection appeared with respiratory symptoms of dyspnoea, pleural and pericardial effusions at autopsy. These adverse events were not observed with the middle dose (5x109 pfu). Therefore it was chosen as the optimal dose. Contrary to the adenoviral vector, plasmid transfer with the dose range used (20–60 µg) did not result in a linear dose–response curve. Due to its highest numerical mean expression, the middle dose (40 µg) was chosen as the optimal dose.

After gene transfer with the optimal doses chosen, hVEGF-A expression following the AdhVEGF-A165 gene transfer was about 20 times higher than following PhVEGF-A165 in the myocardium. This is consistent with previous studies [5,6]. Interestingly, when injected into the peri-infarction region 7 days after the ligation of the coronary artery, both AdhVEGF-A165 and PhVEGF-A165 expressed only about 7% of that expressed in normal myocardium [16]. Reasons for the lower expression might be scar formation with less density of cardiac cells to be transfected by gene therapy. The gene delivered might also have had less spread in the peri-infarction area due to less cardiac systolic function and connective tissue formation after myocardial infarction.

The original hypothesis was that there should be a linear relation between the level of VEGF-A and the degree of angiogenesis. Thus a higher level of VEGF-A from AdhVEGF-A165 transfer should have a more efficient angiogenic effect. Unexpectedly, AdhVEGF-A165 and PhVEGF-A165 induced a similar degree of angiogenesis during the 1-month follow-up.

It appears that the overexpressed hVEGF-A does only have a turn-on or ceiling effect at least with this experimental design with a rat myocardial infarction model. Higher hVEGF-A expression from adenovirus did not have any additive angiogenic effect. Angiogenesis depends on the balance between an array of stimulatory and inhibitory factors [17,18]. It might be that such a balance only permits a small window for hVEGF-A overexpression to stimulate vessel growth. Thus only a small amount of VEGF would be needed to reach saturation of the angiogenesis in the peri-infarction region. Although plasmid expressed less hVEGF-A, the VEGF amount might be sufficient for the angiogenic process.

AdLacZ also induced a transient increment of both capillaries and arterioles after 1 week of treatment. This suggests that the adenoviral infection might cause an inflammatory reaction [19] with secondary transient angiogenesis.

Gene transfer of VEGF induced more pronounced angiogenic effects at the arteriolar level than at the capillary level. This is consistent with previous studies [14,15,20].

Tissue velocity assesses regional myocardial function and perfusion conditions [21,22]. Similar improved ventricular displacement in both AdhVEGF-A165 and PVEGF-A165 suggested that increased mechanical function of the myocardium might be related to the increased perfusion. This indicates that the increased vessels induced by AdhVEGF-A165 and PhVEGF-A165 were functional.

TUNEL stained positive cells were present in low numbers in the non-injected peri-infarction area. A slight increase in number was observed for saline and plasmid injection, indicating little or no apoptotic effect. However, AdLacZ and AdhVEGF-A165 caused a 5 to 8 fold increase in TUNEL stained cells compared with the non-injected area. It suggests that apoptosis might be induced by adenovirus in the injection region.

Adenovirus can modulate apoptosis [8,9]. Adenovirus genes such as E1A and E4orf4 are pro-apoptotic whereas E1B and E3 are apoptosis inhibiting genes. In our experiment, we used the modified first generation adenovirus with deletion of E1 and E3. Still apoptosis was observed. VEGF is known to counteract apoptosis [9,23]. AdhVEGF-A165 showed a non-significant decrease in apoptosis compared with AdLacZ.

We determined apoptosis 7 days after gene transfer. At least some of the apoptotic cells were cardiomyocytes. An impairment of left ventricular function due to the iatrogenic myocardial apoptosis in the ischemic peri-infarction region cannot be excluded in the present study. But in fact, AdLacZ did not show any worsened cardiac function compared with PLacZ. Since adenovirus can induce inflammation [19], the growth factors released by inflammatory cells can induce angiogenesis [24]. Consequently, a transient angiogenic effect was observed in the AdLacZ group. Increased vessel density can increase the perfusion thus might save the ischemic myocardium. Therefore although apoptosis was induced by adenovirus, the side effects might be overwhelmed by the salvage of myocardium induced by increased perfusion.

Ectopic hVEGF-A gene expression in different organs was found to be around 2–5% of that in the myocardium both with adenovirus and plasmid gene transfer. This indicates a similar mechanism of systemic leakage of adenovirus and plasmid vectors from local myocardial delivery. However, AdhVEGF-A165 still induced much higher hVEGF-A expression than plasmid.

In the myocardial infarction model used, the therapeutic window for myocardial angiogenesis appears to be saturated already with the hVEGF-A expression induced by plasmid gene transfer. As adenovirus induces higher local expression of hVEGF-A, it also induces higher systemic expression levels concomitant with potential undesirable effects induced by hVEGF-A as well as tissue inflammation and apoptosis. Thus if there is a therapeutic window with a ceiling effect and AdhVEGF-A165 should be used as angiogenic stimulator, the dose can be reduced to decrease side effects with retained angiogenic effect.

In conclusion, in this myocardial infarction model adenoviral gene transfer has no obvious angiogenic advantage over PhVEGF-A165 but more side effects. This indicates that PhVEGF-A165 might be more applicable than AdhVEGF-A165 for therapeutic angiogenesis.


   Acknowledgements
 
This study was supported by grants from the Swedish Medical Research Council (9515), the Swedish Heart and Lung Foundation, the Karolinska Institute Foundations and the Belvén Foundation. The adenoviral vectors were a generous gift from Seppo Ylä-Herttuala, Department of Medicine A.I.Virtanen Institute, Kuopio, Finland.


   Notes
 
Time for primary review 28 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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