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(Investigative Ophthalmology and Visual Science. 2000;41:4268-4274.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Incorporation and Differentiation of Hippocampus-Derived Neural Stem Cells Transplanted in Injured Adult Rat Retina

Akihiro Nishida1,2, Masayo Takahashi1, Hidenobu Tanihara1, Ichiro Nakano3, Jun B. Takahashi3, Akira Mizoguchi2, Chizuka Ide2 and Yoshihito Honda1

1 From the Departments of Ophthalmology and Visual Sciences, 2 Anatomy and Neurobiology, and 3 Neurosurgery, Graduate School of Medicine, Kyoto University, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
PURPOSE. In a previous study it has been shown that adult rat hippocampus-derived neural stem cells can be successfully transplanted into neonatal retinas, where they differentiate into neurons and glia, but they cannot be transplanted into adult retinas. In the current study, the effect of mechanical injury to the adult retina on the survival and differentiation of the grafted hippocampal stem cells was determined.

METHODS. Mechanical injury was induced in the adult rat retina by a hooked needle. A cell suspension (containing 90,000 neural stem cells) was slowly injected into the vitreous space. The specimens were processed for immunohistochemical studies at 1, 2, and 4 weeks after the transplantation.

RESULTS. In the best case, incorporation of grafted stem cells was seen in 50% of the injured retinas. Most of these cells located from the ganglion cell layer through the inner nuclear layer close to the injury site. Immunohistochemically, at 1 week, more than half of the grafted cells expressed nestin. At 4 weeks, some grafted cells showed immunoreactivity for microtubule-associated protein (MAP) 2ab, MAP5, and glial fibrillary acidic protein (GFAP), suggesting progress in differentiation into cells of neuronal and astroglial lineages. However, they showed no immunoreactivity for HPC-1, calbindin, and rhodopsin, which suggests that they did not differentiate into mature retinal neurons. Immunoelectron microscopy revealed the formation of synapse-like structures between graft and host cells.

CONCLUSIONS. By the manipulation of mechanical injury, the incorporation and subsequent differentiation of the grafted stem cells into neuronal and glial lineage, including the formation of synapse-like structures, can be achieved, even in the adult rat retina.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Since the mid-1990s, it has been possible to isolate neural stem or progenitor cells from various parts of the central nervous system (CNS), such as the hippocampus, subventricular zone, spinal cord, and ependyma.1 2 3 4 In general, these cells can expand in serum-free medium and proliferate in response to growth factors such as epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF). From a clinical point of view, they have some potential advantages for retinal transplantation compared with embryonic or newborn retinal cells. First, they can be expanded through numerous passages in vitro and frozen for storage. Second, they can be easily manipulated, such as by pretreatment with growth factors or gene transduction, before they are transplanted.

Adult rat hippocampus-derived neural stem cells, first isolated by Palmer et al.5 , are one of the few cell lines that have been shown by clonal analysis to have multipotency and self-renewability. In a previous study of ours, we found that the hippocampal stem cells could be successfully transplanted and integrated into the neonatal rat retina but that when they were transplanted into adult eyes, they aggregated on the surface but never migrated into the retina.6

In this study, for the purpose of assessing the possibility and limitations of the use of brain-derived neural stem cells for retinal transplantation, we investigated whether these hippocampal stem cells could migrate and become incorporated into mechanically injured adult rat retinas.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusions
 References
 
Preparation of Cells for Grafting
LacZ-labeled clonal adult rat hippocampus-derived neural stem cells (clone PZ5, kindly provided by Fred H. Gage, Salk Institute, La Jolla, CA) were used in this study. They were cultured on laminin/poly-L-ornithine–coated dishes containing Dulbecco’s modified Eagles medium-Ham’s F12 (DMEM-F12; Gibco, Rockville, MD) supplemented with N2 (Gibco) and 20 ng/ml bFGF (Genzyme, Cambridge, MA), and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. After having been subcultured for 2 weeks to 3 months, they were harvested for grafting with 0.05% trypsin in DMEM-F12, washed with 0.01% trypsin inhibitor (Wako, Osaka, Japan) in DMEM-F12, and suspended at a density of 30,000 cells/µl in high-glucose Dulbecco’s phosphate-buffered saline (D-PBS, Gibco) containing 20 ng/ml bFGF.

Animal Preparation and Grafting Procedure
Eight-week-old male Fischer rats (n = 30) were obtained from Shimizu Laboratory Supplies (Kyoto, Japan). All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The animals were anesthetized with a mixture (1:1) of xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride (10 mg/kg) administered intramuscularly. The pupils were dilated with 0.5% tropicamide and 2.5% phenylephrine eye drops. The corneas were anesthetized with drops of 0.4% oxybuprocaine hydrochloride. The eyeballs were perforated at the equator with a 27-gauge needle. A hooked 30-gauge needle was then inserted through the wound, and the retina was injured by scratching it parallel to the equator between the retinal vessels under direct observation with a surgical microscope equipped with a plano-concave contact lens for rats (Kyocon, Kyoto, Japan). Special care was taken to injure the whole layer of the retina, and success was affirmed by a small amount of subretinal bleeding. After the injury, 3 µl of the cell suspension (containing 90,000 cells) was slowly injected into the intravitreal space with a microsyringe fitted with a 30-gauge blunt needle (15 rats, 30 eyes). As a control, 3 µl of the cell suspension was injected into the intravitreal space of noninjured eyes (15 rats, 30 eyes). The results from five eyes of the control group were excluded due to complications of massive vitreous hemorrhage.

Tissue Sectioning
The animals were anesthetized by inhalation of diethyl ether and fixed by transcardial perfusion with 4% paraformaldehyde (Merck, Darmstadt, Germany) in 0.1 M phosphate buffer (PB) 1, 2, and 4 weeks later. The eyes were enucleated to make eyecups. The eyecups were immersed in the same fixative for 2 hours at 4°C and then in 15%, 20%, and 25% sucrose-PBS for cryoprotection. They were embedded in optimal cutting temperature compound (OCT; Miles, Elkhart, IN) after adjustment of their horizontal planes parallel to the cutting plane, and 20-µm frozen sections were made in a cryostat. Continuous sections including the injury site were cut for each eye.

Immunocytochemistry
The specimens were washed with 0.1 M PB and then incubated with 20% skim milk (Dainihon–Seiyaku, Osaka, Japan) in 0.1 M PB containing 0.005% saponin (0.1 M PB-saponin; Merck) for 10 minutes to block nonspecific antibody binding. They were then incubated with primary antibodies diluted in 5% skim milk in 0.1 M PB-saponin for 24 hours at 4°C. Antibodies and concentrations used in this study were as follows: mouse monoclonal anti-ß-galactosidase (ß-gal, 1:1000; Promega, Madison, WI), rabbit polyclonal anti-ß-gal (1:5000; Chemicon, Temecula, CA), mouse monoclonal anti-nestin (1:1000; PharMingen, San Diego, CA), mouse monoclonal anti-microtubule associated protein (MAP) 2ab (1:100; Sigma, St. Louis, MO), mouse monoclonal anti-MAP5 (1:1000; Chemicon), rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; 1:1000; Chemicon), rabbit anti-myelin basic protein (MBP; 1:500; UltraClone, Wellow, UK), mouse monoclonal anti-HPC-1 (1:1000; Sigma), mouse monoclonal anti-calbindin (1:500; Sigma), and rabbit anti-rhodopsin (1:1000; LSL, Tokyo, Japan).

After the reaction with primary antibodies, the specimens were washed with 0.1 M PB-saponin and incubated with secondary antibodies diluted in 5% skim milk in 0.1 M PB-saponin for 90 minutes. Antibodies and concentrations used in this study were as follows: fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse immunoglobulin (1:100; Amersham, Buckinghamshire, UK), FITC-conjugated donkey anti-rabbit immunoglobulin (1:100; Amersham), Cy5-conjugated goat anti-mouse IgG (1:200; Amersham), and Cy5-conjugated donkey anti-rabbit IgG (1:200; Amersham).

Sections were then washed with 0.1 M PB, mounted with glycerol-PBS (1:1) and observed with a laser-scanning confocal microscope (1024; Bio-Rad, Hercules, CA).

Immunoelectron Microscopy
Immunoelectron microscopy using the silver-enhancement technique was done as described.7 Briefly, after having been blocked with 20% skim milk in 0.1 M PB-saponin, the sections were incubated with the anti-ß-gal antibody (1:1000; Promega) and subsequently with an anti-mouse IgG antibody coupled with 1.4-nm gold particles (1:50; Nanoprobes, Stony Brook, NY). After the sections had been washed, they were fixed with 1% glutaraldehyde (Nacalai Tesque, Kyoto, Japan) in 0.1 M PB for 10 minutes, and the sample-bound gold particles were then silver-enhanced at 20°C for 12 minutes by use of an HQ-silver kit (Nanoprobes). They were again washed and postfixed with 0.5% osmium oxide (Nacalai Tesque) in 0.1 M PB at pH 7.3, dehydrated by passage through a graded series of ethanol (50%, 60%, 70%, 80%, 90%, 95%, and 100%), and embedded in epoxy resin. From these samples, ultrathin sections were cut, stained with uranyl acetate and lead citrate, and then observed with an electron microscope (JEM-1200EX; JEOL, Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusions
 References
 
Incorporation and Distribution of Grafted Cells
In an attempt to elucidate the efficacy of transplantation of hippocampal stem cells into the adult rat retina, we injected them into the vitreous space. The stem cells were labeled with the LacZ gene retrovirally, so that we could identify ß-gal–immunoreactive cells as the grafted cells. In our previous study, we confirmed that ß-gal enzyme leaking from damaged or dead grafted cells was not taken up by host retinal cells.6

First, we compared the incidence of eyes with incorporated grafted cells between the injured group and the noninjured group. In the injured group, 1 week after transplantation, ß-gal–immunoreactive cells were incorporated into the host retina in 10% of the experimental eyes (1 of 10). At 2 and 4 weeks, the percentage of eyes with incorporated cells increased to 50% (5 of 10) and 40% (4 of 10), respectively (Table 1) . In the eyes with incorporated grafted cells, the grafted cells were distributed around the site of injury, where GFAP immunoreactivity of the host retina was upregulated (Fig. 1A ). In contrast, no eyes incorporated grafted cells in the noninjured group at any period after transplantation (Table 1) . The grafted cells were found to have aggregated on the inner surface but never to have been incorporated into the host retina of the noninjured group (Fig. 1B) . Statistical analysis by Fisher’s exact probability test showed a significant difference (P < 0.05) between the injured and noninjured groups in the incidence of successful incorporation of the grafted cells at both 2 and 4 weeks after transplantation.


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  		Table 1. Incidence of Eyes with Incorporated Grafted Cells

 


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  		Figure 1. Double-label immunofluorescence study using antibodies against ß-gal (green) and GFAP (red) in the injured group (A) and noninjured group (B) 2 weeks after the injection. (A) ß-Gal–positive grafted cells were observed primarily in the GCL and INL in the host retina around the site of injury (arrow). Expression of GFAP in the host retina was upregulated. (B) ß-Gal–positive cells were located on the inner surface of but not within the host retina. The expression of GFAP was localized in the astrocytes and the end feet of the Müller cells. Scale bar, 100 µm.

 
The pattern of grafted cell distribution was almost the same at all times after the injection. The grafted cells were observed not only at the site of injury where normal retinal structure was destroyed, but also in the surrounding area where the normal retinal structure was retained. Most of them were situated in the inner nuclear layer (INL) with some in the ganglion cell layer (GCL), where they formed a layer-like structure. A few grafted cells were found on the inner surface of the retina and in the outer nuclear layer (ONL). The width of distribution of the incorporated grafted cells ranged between 790 µm and 1200 µm around the site of injury (data not shown). This width was much greater than that of the actual injury in all cases, which was less than 100 µm.

The grafted cells adherent to the inner surface of the host retina in the injured group were round and had no processes, whereas most incorporated cells had elongated processes, and some of them showed morphologies reminiscent of amacrine and bipolar cells (Fig. 2) .



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  		Figure 2. ß-Gal–immunoreactive grafted cells, which are similar to amacrine (A, arrow) and bipolar (B, arrow) cells, 1 and 4 weeks after transplantation, respectively. Scale bar, 20 µm.

 
Immunohistochemistry on Sections after Transplantation
Immunohistochemical studies were performed on sections with incorporated grafted cells in the injured group. The sections were double immunostained with anti-ß-gal antibody and antibodies against specific cell-type markers. The cell-type markers used were nestin for immature or undifferentiated cells, MAPs for neuronal lineage cells, GFAP for astrocytes and Müller cells, MBP for oligodendrocytes, HPC-1 for amacrine cells, calbindin for horizontal and some amacrine cells, and rhodopsin for rod photoreceptor cells. The ratios of double-stained cells to ß-gal–positive cells were calculated to estimate the characteristics of the grafted cells after transplantation.

Our preliminary studies showed the presence of nestin immunoreactivity in more than 96% of the cultured hippocampal stem cells; however, no immunoreactivity for other specific markers of differentiated cell types, including MAP2ab, MAP5, GFAP, MBP, HPC-1, calbindin, and rhodopsin, was detected (data not shown).

Among the grafted cells, nestin-positive cells were over 50% at the end of 1 and 2 weeks after transplantation; however, they decreased to 36% after 4 weeks (Table 2 , Figs. 3A 3B 3C ). MAP5-positive cells increased markedly from 1% to 22% between 1 and 2 weeks, whereas MAP2ab-positive cells gradually increased from 1 to 4 weeks (Table 2 , Figs. 3D 3E 3F ). As for the two glial markers, GFAP-positive grafted cells increased from 2% to 10% between 2 and 4 weeks, but MBP-positive cells were hardly observed from weeks 1 through 4 (Table 2 , Figs. 3G 3H 3I ). Immunoreactivity for retinal cell markers, HPC-1, calbindin, and rhodopsin was hardly detected in the grafted cells throughout the 4 weeks (Table 2 , Figs. 3J 3K 3L ).


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  		Table 2. Differentiation Ratio of the Incorporated Grafted Cells in the Injured Group

 


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  		Figure 3. Double-label immunofluorescence at the end of 1 (A, D, G, and J), 2 (B, E, H, and K), and 4 (C, F, I, and L) weeks after cell transplantation. Green: ß-Gal–immunoreactive cells; red: nestin- (A, B, and C), MAP5- (D, E, and F), GFAP- (G, H, and I), and calbindin- (J, K, and L) immunoreactive cells; yellow: double-stained cells (arrows). (A, B, and C) Nestin-positive grafted cells decreased in number from 1 to 4 weeks after transplantation. (D, E, and F) MAP5-positive grafted cells increased from 1 to 4 weeks after transplantation. (G, H, and I) Few GFAP-positive grafted cells are observed at 1 and 2 weeks after transplantation, but they begin to appear at 4 weeks. (J, K, and L) Calbindin-positive grafted cells are rarely observed at any time after the injection. Scale bar, 20 µm.

 
The immunoreactivity for nestin and GFAP was also observed in the host Müller cells around the sites of injury, where the grafted cells were incorporated into the host retinas (Figs. 3A 3B 3C 3G 3H 3I) .

Immunoelectron Microscopy on Sections at 4 Weeks after Transplantation
Immunoelectron microscopy was performed on sections of 4-week specimens. Grafted cells were identified by the presence of gold particles indicating immunoreactivity for ß-gal. In general, the grafted cells had heterochromatic nuclei and a large number of mitochondria (Figs. 4A 4B ).



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  		Figure 4. Immunoelectron microscopy on sections at 4 weeks after transplantation. (A) Grafted cells are identified by the presence of gold particles indicating immunoreactivity for ß-gal. A gold-labeled grafted cell (G1) in the IPL extended its pseudopodia (p) and made contact with another grafted cell (G2). Note that the grafted cells contained a large number of mitochondria (mt). (B) A grafted cell (G) extended its process (arrow) and made close contact with a host cell (H) in the innermost part of the INL. (C) A grafted cell (G) in the INL formed contacts with host cells (H). Both symmetrical (arrows) and asymmetrical (arrowhead) membrane thickenings were observed. (D) An axon terminal of a grafted cell (G) labeled with gold particles (small arrows) in the IPL contained synaptic vesicles (arrowheads) and formed a synapse-like structure with a host cell (H). Postsynaptic density (large arrow) was observed in the host cell. Scale bar: 1 µm (A, B); 500 nm (C, D).

 
In the inner plexiform layer (IPL) and at the innermost part of the INL, grafted cells were often found in a group (Fig. 4A) . Some of them were irregular in shape and had pseudopodia that made contact with other grafted cells (Fig. 4A) , which is a characteristic of actively migrating cells. Some other grafted cells had a relatively round shape and extended their processes to make close contact with host cells at the innermost part of the INL (Fig. 4B) . At a higher magnification, symmetrical and asymmetrical membrane thickening, which represent puncta adherentia and synaptic junctions, respectively, were observed between graft and host cells (Figs. 4C 4D) indicating that they formed close contacts with each other.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusions
 References
 
Neural stem cells are expected to be useful clinically for replacing damaged neurons or for ex vivo gene therapy.8 In the field of brain science, they have been tested on damaged brain models9 10 as cell resources for replacement therapy. Also in the field of ophthalmology, it is reported that neural stem cells could be successfully transplanted into damaged retina.11 12 13 Therefore, it is important to assess the application of neural stem cells for retinal transplantation therapy.

This study has shown the ability of hippocampus-derived neural stem cells to migrate and differentiate in the injured retina. However, the limitation of their differentiation into authentic retinal neurons was also recognized.

Pattern of Incorporated Grafted Cells in the Host Retina
The incidence of the eyes with incorporated grafted cells increased between 1 and 2 weeks but did not change between 2 and 4 weeks. Some time may be required for the cells that have migrated onto the retinal surface to create graft–host contacts and to migrate into the host retina. This behavior of the grafted cells is consistent with the results of our previous study.6

The grafted cells were located around the injured sites, where the expression of nestin and GFAP in the host Müller cells was upregulated. The width of the distribution of the grafted cells was much greater than that of the injury (less than 100 µm) at any time point evaluated. We therefore speculate that the grafted cells migrated into the host retina not only from the injured site but also from the vitreous surface around the injured site where the host Müller cells were activated by the injury. This speculation was supported by our other experiments that hippocampal stem cells can also incorporate into chemically damaged retinas (data not shown). It has been reported that upregulation of the expression of nestin and GFAP in astrocytes or Müller cells occurred in the CNS including the retina after various types of damage.14 15 16 17 18 It also has been shown that activated Müller cells express a number of cytokines such as bFGF, ciliary neurotrophic factor (CNTF), and transforming growth factor (TGF)-{alpha}.19 20 21 22 It seems reasonable that the Müller cells that were activated by the mechanical injury may have played an important role in the migration and/or differentiation of the surviving grafted cells.

For the purpose of assessing the effect of retinal injury, we chose the vitreous cavity instead of the subretinal space for the site of injection of the neural stem cells. Subretinal injection itself causes retinal detachment and much damage to the retina.

Differentiation and Integration of the Grafted Cells
The hippocampal stem cells used as the grafted cells were confirmed by immunocytochemistry to be immature cells. Before grafting, most of them expressed nestin. However, once they were grafted, the number of cells expressing nestin decreased. On the contrary, the cells expressing MAPs and GFAP increased with time, which suggests differentiation of the stem cells into cells of the neuronal and astroglial lineages. Among the MAPs, MAP2ab is thought to be a late marker of neuronal differentiation, because its expression increases as neuronal cells mature,23 whereas the expression of MAP5 is generally abundant in neuronal cells at very early developmental stages.23 These facts explain why the expression of MAP5 in the grafted cells increased earlier than that of MAP2ab. GFAP and MBP are markers for astrocytes and oligodendrocytes, respectively. The expression of MBP was hardly detected up to 4 weeks, whereas that of GFAP increased between 2 and 4 weeks after the grafting. This finding indicates that the hippocampal stem cells did not differentiate into oligodendrocytes but into astrocytes after the grafting, although they differentiated into both glial lineages in vitro.5 It also suggests that the specific microenvironment in the retina, where no oligodendrocytes exist, may affect the fate of differentiation of the hippocampal stem cells. As for the retinal cell markers, HPC-1, calbindin, and rhodopsin, their expression in the grafted cells was hardly observed at any time after the grafting, indicating the failure of differentiation into retinal neurons even at the end of 4 weeks after the grafting. One possible reason for the failure is absence of unknown local cues in injured adult retina. There may be some unknown factors that are expressed only in earlier stages of retinal development and permit the hippocampal stem cells to differentiate into retinal neurons. Another possible explanation is limited plasticity of the hippocampus-derived neural stem cells. They may continue to possess the characteristics of cells in the hippocampus, from which they are derived, even after being transplanted into retinal tissue.

Immunoelectron microscopic study revealed the existence of graft–graft and graft–host contacts. The grafted cells formed puncta adhaerentia-like and asymmetrical synapse-like structures with the host cells. Not only mechanical contacts but also intercellular signaling could be formed between the graft and host cells. There are several reports describing graft–host synapse formation in the adult CNS in homotopic transplantation, such as retina to retina,24 25 and also in heterotopic transplantation, such as retina to cerebellum.26 It is still unknown whether these synapse-like structures actually function; however, the formation of such structures is significant evidence for integration of the grafted cells into the host retina.

Deriving Retinal Neurons from Neural Stem Cells
Further studies are needed to establish the utility of neural stem cells for replacement and reconstruction of retinal neurons. One possibility is retina-derived neural progenitor cells. A recent study revealed that embryonic retina-derived neural progenitor cells can differentiate into photoreceptors in vitro.27 If they maintain the characteristics of retinal cells through expansion in vitro, they may differentiate into retina-specific neurons after transplantation. Another possibility is modification of cellular characteristics of the hippocampus-derived neural stem cells for retina-specific differentiation by transfection of key molecules such as homeobox genes.28 29 Also, pretreatment of the neural stem cells with growth factors is a possible means of controlling the cells’ fate. In fact, in our previous study, we found that some neurotrophins affect the differentiation of the hippocampal stem cells in vitro30 ; however, growth factors that can induce neural progenitor cells to produce retina-specific neurons have not yet been identified.


   Conclusions
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
References
 
In conclusion, this study has yielded basic and important information regarding the transplantation of adult rat hippocampus-derived neural stem cells into the adult retina. First, incorporation of the grafted neural stem cells was achieved in injured adult retinas. Second, some of the incorporated neural stem cells showed differentiation into neuronal lineage and formed graft–host contacts such as puncta adhaerentia- and synapse-like structures. Third, even after successful transplantation and differentiation into cells of the neuronal lineage, the neural stem cells failed to differentiate into retina-specific phenotypes as shown by expression of HPC-1, calbindin, and rhodopsin, possibly because of their basic inability or an absence of local cues essential for differentiation into retinal neurons.


   Acknowledgements
 
The authors thank Fred H. Gage at the Salk Institute for helpful comments.


   Footnotes
 
Supported in part by a Grant-in-Aid and Health Science Research Grants for Research on Brain Science from the Ministry of Health and Welfare of Japan, by grants from the Japan Society for the Promotion of Science and the Japan National Society for the Prevention of Blindness, and by Grants-in-Aid 090280101, 10897014, and 10044272 from the Ministry of Education, Science, Sports and Culture of Japan.

Submitted for publication May 22, 2000; revised July 5, 2000; accepted July 25, 2000.

Commercial relationships policy: N.

Corresponding author: Masayo Takahashi, Department of Ophthalmology and Visual Sciences, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kawaharacho, Shogoin, Kyoto 606-8507, Japan. masataka{at}kuhp.kyoto-u.ac.jp


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 

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