Platelet lysates stimulate angiogenesis, neurogenesis and neuroprotection after stroke

© Schattauer 2013 Thrombosis and Haemostasis 110.2/2013

323Platelets and Blood Cells

Platelet lysates stimulate angiogenesis, neurogenesis and neuroprotection after strokeYael Hayon1; Olga Dashevsky2; Ela Shai2; David Varon2; Ronen R. Leker1

1Department of Neurology, Peritz and Chantal Scheinberg Cerebrovascular Research Laboratory, Hadassah Ein Kerem, Jerusalem, Israel; 2Department of Hematology, Coagulation Unit Hadassah University Hospital, Hadassah Ein Kerem, Jerusalem, Israel

SummaryPlatelets contain chemo-attractants and mitogens that have a major role in tissue repair. Therefore we hypothesised that tissue regener-ation secondary to activation of endogenous neural stem cells (eNSC) can be enhanced by delivering platelets to the ischaemic brain. To examine these potential therapeutic effects we injected platelet-poor plasma (PPP), fibroblast growth factor (FGF2) and platelet lysate (PLT) to the lateral ventricles after permanent middle cerebral artery occlu-sion (PMCAO) in rats. The animals were tested with the neurological severity score, and infarct volumes were measured at 90 days post–PMCAO. Immunohistochemistry was used to determine the fate of newborn cells and to count blood vessels in the ischaemic brain.

Platelets significantly increased eNSC proliferation and angiogenesis in the subventricular zone (SVZ) and in the peri-lesion cortex. Func-tional outcome was significantly improved and injury size was signifi-cantly reduced in rats treated with PLT suggesting additional neur-oprotective effects. In conclusion, local delivery of PLT to the lateral ventricles induces angiogenesis, neurogenesis and neuroprotection and reduces behavioural deficits after brain ischaemia.

KeywordsAngiogenesis, neurogenesis, neuroprotection, platelets, cerebral ischaemia

Correspondence to:R.R. Leker, MDStroke Service and the Peritz and Chantal Cerebrovascular Research LaboratoryHadassah Ein Kerem P. O. Box 12000, Jerusalem 91120, IsraelTel.: +972 2 677 6945, Fax: +972 2 643 7782 E-mail: [email protected] Varon, MDCoagulation Unit Hadassah Ein Kerem P. O. Box 12000, Jerusalem 91120, IsraelTel.: +972 2 677 7672, Fax: +972 2 644 9580 E-mail: [email protected]

Financial support: This study was funded by Ministry of Science, Israel.

Received: November 28, 2012 Accepted after major revision: May 2, 2013 Prepublished online: June 13, 2013

doi:10.1160/TH12-11-0875Thromb Haemost 2013; 110: 323–330

Introduction

Despite major medical advances, stroke is still a leading cause of death and disability worldwide (1). Activation of endogenous neu-ral stem cells (eNSC) has been proposed as a novel form of therapy in a variety of neurological disorders, including stroke. Previous data suggest that application of external factors can boost long-term endogenous repair mechanisms in the cerebral cortex (2, 3).

Platelets contain a plethora of bioactive components that can be released under various physiologic and pathologic conditions. Per-tinent examples include platelet-derived growth factor (PDGF), fi-broblast growth factor (FGF), transforming growth factor β (TGFβ), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and insulin growth factor (IGF) (4). Recent data suggests that platelets have broad therapeutic applications in-cluding treatment of ischaemic ulcers and enhancement of wound healing (5). In addition, because of the proliferative effect of pla-telet-derived factors on different cell types, platelet-derived factors could potentially lead to expansion of tissue specific stem cell populations including eNSC (6).

In the adult brain, the subventricular zone (SVZ) contains a stem cell niche that is rich in blood vessels. These neural precur-sors have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes and can take part in cortical repair after stroke (3, 7-10). We and others have shown that platelet microparticles (PMP) promote all stages of angiogenesis in vivo in addition to pro-coagulant and inflammatory effects, and are capable of im-proving reperfusion in a rat myocardial infarction model (11, 12).

We and others have also previously shown that incubation with PMP in vitro increases angiogenesis as well as survival, prolifer-ation and differentiation of neural stem cells (11, 13, 14) in a mechanism that involves signalling through pAKT and pERK (13). These effects were also shown in vivo in a model of cerebral ischaemia where PMP were applied topically on the injured brain using a biodegradable polymer (10).

In the current study we wished to examine the effects of the parent cell; the platelets, on eNSC and on angiogenesis using a dif-ferent application method of direct intra-cerebro-ventricular (ICV) injection. Herein we show that lysates made from platelets support the neurovascular niche and significantly improve motor

For personal or educational use only. No other uses without permission. All rights reserved.Downloaded from www.thrombosis-online.com on 2013-12-02 | ID: 1000491814 | IP: 217.110.19.91

Thrombosis and Haemostasis 110.2/2013 © Schattauer 2013

324 Hayon et al. Delivery of therapeutic agents after cerebral ischaemia

function with a distinct decrease in infarct volume after cerebral ischaemia.

Materials and methodsPlatelet lysates preparation

Platelets were isolated from healthy volunteers according to insti-tutional Blood Bank regulations and with the approval of the insti-tutional ethics committee. Fresh platelet-rich plasma (PRP) was prepared by drawing 10 ml of blood in citrate tubes and centrifu-gation of the whole blood at 120 x g for 12 minutes (min). PRP was diluted in PBS (1:5; without Ca++, Mg++). Next, PRP was centri-fuged (750 x g 5 min), the plasma was removed and the platelet pellet was resuspended in phosphate-buffered saline (PBS) (with-out Ca++, Mg++). Finally, platelets were frozen (-80°C) and thawed (37°C) three times and then used in the current experiment.

Animals

Male spontaneously hypertensive rats (SHR; 13 weeks old; n=9/group) were housed in standard conditions (under a 12 hours light/dark cycle), food and water were available ad libitum at all times. This study was carried out in strict accordance with the rec-ommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the institutional Animal Use and Care Committee.

Permanent middle cerebral artery occlusion

Focal ischaemia was produced by permanent distal middle cer-ebral artery occlusion (PMCAO) as previously described (10). A sham operated group was not included in the current set of experi-

ments as sham animals do not develop any deficits (10). Immedi-ately following ischaemic brain injury animals were injected with platelet-poor plasma (PPP; negative control), fibroblast growth factor (FGF, 0.1 µg/ml positive control) - or platelet lysate (PLT, 100 µg/ml) to the lateral ventricle. By using this application tech-nique we aimed to generate the potential regenerative effect, while avoiding the pro-thrombotic risk of delivering platelets and their products into the blood circulation. Platelet lysate dose was based on unpublished in-vitro data and platelet microparticles dosage from previous publication (10).

BrdU injections

All animals received 5-bromo-2-deoxyuridine (BrdU; 50 mg/kg IP bid) to label dividing cells on days 1-7 post-surgery.

Neurobehavioural evaluation

Animals were examined with a standardised neurologic severity score during three months after injury. Ten different tasks were used to evaluate motor ability, balancing, and alertness of the tested ani-mal and points were given for failure to perform a task (15).

Injury size

At 90 days after the surgery the animals were deeply anesthetised and perfusion fixed with 4% paraformaldehyde. Brains were frozen-sectioned at 10 µm. Brain slices 200 µm apart between bregma +1.42 and bregma -0.8 were stained with Giemsa stain modified solution (Fluka, Sigma-Aldrich, St. Louis, MO, USA) and digitally photographed. The volume of injured tissue was measured with image J software (National Institutes of Health, Be-thesda, MD, USA). Damaged tissue volume was calculated by di-

Figure 1: Platelet lysate improve behaviou-ral deficits after stroke. Animals (n=9/group) underwent permanent middle cerebral artery occlusion (PMCAO) were treated with platelet-poor plasma (PPP), fibroblast growth factor (FGF) and platelet lysate (PLT). Neurologic dis-ability was evaluated at different time points after PMCAO with the neurologic severity score (NSS). Animals treated with PLT showed signifi-cantly larger improvement beginning at day 7 after PMCAO, and the curves continued to di-verge until the end of the study. Note that motor functions are much better in the PLT treated animals compared to vehicle and FGF treated animals. Data are represented as mean ± SEM, * p<0.05.



325Hayon et al. Delivery of therapeutic agents after cerebral ischaemia

viding the volume of the injured hemisphere by that of the non-lesioned hemisphere (16).

The results are expressed as a percentage of hemispheric tissue:

([contralateral – ipsilateral]/contra)*100 = lesion volume

Immunohistochemistry

At day 90 post injury, brains were perfusion fixed and frozen sec-tions were prepared at 10 µm. Brain slices were double or triple stained for immunohistochemical evaluation using fate specific antibodies that included rat anti BrdU (Accurate, Westbury, NY, USA; 1:200), rabbit anti Sox2 and rabbit anti Nestin (Chemicon, Millipore, Billerica, MA, USA; 1:200), rabbit anti GFAP (Dako, Glostrup, Denmark; 1:200), mouse anti NeuN (Chemicon; 1:200), and mouse anti RECA1 (Serotec, Düsseldorf, Germany; 1:200). Alexa 488 and Alexa 555 conjugates were used as secondary anti-bodies (Molecular Probes, Leiden, The Netherlands; 1:200), and DAPI (Sigma, Jerusalem, Israel) was used to visualise nuclei.

Cell counting

Immuno-positive cells were counted using an epifluorescent Olympus microscope in pre-specified regions of interest (ROI) in-cluding the SVZ and the areas surrounding the lesioned cortex.

Cells were counted in a semi-quantitative manner on high power field magnifications (X400). Specifically, we studied equidistant slices, 100 µm apart, from bregma +1.42 to bregma -0.8. In each slice, cells were counted in 10 equidistant fields per ROI (30 fields per slide and 360 fields per brain at X400 magnification).

Statistical analysis

All experiments were done according to STAIR recommendations. Clinical and immunohistochemical evaluations were performed by an examiner that was, blinded to the experimental group. Analysis was performed with the SigmaStat software package (Sys-tat, Richmond, CA, USA). Data are presented as mean ± SE as in-dicated in the legends. Values were compared using one-way analysis of variance followed by Bonferroni correction for multiple comparisons. P-values < 0.05 were considered significant for all comparisons.

ResultsPlatelet lysate significantly increase functional gain and decreases lesion volume after stroke

Neurological deficits were assessed at predetermined time points after PMCAO using the neurological severity score (NSS). A more

Figure 2: Platelet lysate decrease infarct size after stroke. Equidistant brain slices were stained with Giemsa and the volume of infarct lesion was measured (a). b) Bar graph showing infarct volumes in the different treatment groups (n=9/group) as measured at 90 days after permanent middle cerebral artery occlusion (PMCAO). Animals treated with platelet lysate (PLT) had significantly smaller lesions at each level. Note that infarct volumes are much smaller in the PLT treated animals compared to vehicle and FGF treated animals. Data are repre-sented as mean ± SEM, * p<0.05.

a

b




rapid and pronounced improvement in NSS was observed in ani-mals treated with PLT, as compared with vehicle (PPP) and FGF2-treated animals (▶ Figure 1; n=9/group). The differences in NSS in the PLT-treated animals were apparent as early as day 7 after PMCAO suggesting an early neuroprotective effect. The dif-ference continued to increase with time and remained significantly different until the conclusion of the study in all treatment groups (▶ Figure 1).

Of note, the slope of NSS change with time significantly in-creased after 10 days, and the plot diverged significantly more from vehicle-treated animals in the PLT-treated animals at this time point similar to what was seen after cerebral ischaemia (17).

A significant reduction in lesion size was identified at 90 days after PMCAO in PLT-treated animals (~2-fold decrease compared with PPP-treated animals), supporting a neuroprotective effect for this treatment (▶ Figure 2).

Of note, the behavioural and the neuroprotective effects of pla-telets were significantly larger than the effects of FGF2 alone.

Platelet lysate significantly increase the number of blood vessels in the affected hemisphere after stroke

At least part of the benefit afforded by platelets could be attributed to their well-known effects on angiogenesis (10, 18). To examine the effect of exogenous PPP, FGF2 and PLT on angiogenesis, blood vessel density in the hemisphere ipsilateral to the stroke was evalu-ated in all animals (n=9/group) at the infarct border zone using an antibody against the endothelial marker RECA1 (▶ Figure 3 a). A significant increase in the number of blood vessels was observed in the cortex around the lesion in all treated animals (▶ Figure 3 b), but the largest effects seen in animals treated with PLT (~3-fold in-crease compared with PPP-treated animals), suggesting that treat-ment either protected existing blood vessels or led to a pro-angio-genic effect resulting in the formation of new blood vessels. Of note, the effect of platelets was significantly larger than the effects of FGF2 alone.

Figure 3: Platelet lysate increase angiogenesis in the ischaemic hemisphere. Immunohistochemistry for blood vessels was evaluated at the infarct border. Blood vessel density was counted at the peri-infarct areas (n=9/group). a) Low power photomicrograph (x200) from the infracted hemi-sphere of vehicle treated animals, FGF treated animals and platelet lysate

(PLT) treated animals stained with an antibody to RECA1. b) Bar graph show-ing blood vessel density in the tissue adjacent to the infarct. Note that blood vessel density is much higher in the PLT treated animals compared to vehicle and FGF treated animals. HPF = high power field, Bar graphs 50 µm. Data are represented as mean ± SEM, * p<0.05.

a

b




Platelet lysate significantly increase the number of proliferating cells after stroke

To assess the effect of platelets on cell proliferation, we measured the total number of newborn cells at 90 days after PMCAO. Ani-mals (n=9/group) treated with PLT showed a significant increased in the number of BrdU positive cells at the SVZ (~2-fold increase compared with PPP-treated animals), and at the infarct border zone (~1.5-fold increase compared with PPP-treated animals) (▶ Figure 4 a). Most of these cells were localised to the immediate peri-lesion area (▶ Figure 4 b), suggesting accumulation of prolif-erating cells around the injured brain area. These results suggest a possible mitogenic or pro-survival effect of platelets on dividing cells, which may also promote migration towards the infarct zone. We did not observe any excess of apoptotic death in the brains of the treated animals.

Platelet lysate significantly increase gliogenesis and neurogenesis after stroke

To determine the fates of newborn cells after PMCAO, we used immunohistochemical methods with double and triple staining. A large percentage of newborn cells at the immediate infarct border remained undifferentiated (▶ Figure 5 b, Nestin+) in all animals (n=9/group). Nevertheless, the number of these undifferentiated cells was significantly larger in PLT-treated animals (~2-fold in-crease compared with PPP-treated animals). Most newborn cells that differentiated into mature neurons (▶ Figure5 a, NeuN+) or into astrocytes were located around the injured cortex, suggesting site-appropriate differentiation of the newborn cells.

Overall, the total numbers of BrdU+ neurons and astrocytes at the infarct border were significantly larger in PLT-treated animals (3.5- and 2.5-fold increase compared with PPP-treated animals, re-spectively, ▶ Figure 5 b).

Of note, the effect of PLT was significantly larger than the ef-fects of FGF2 alone.

Figure 4: Platelet lysate increase cell proliferation after stroke. The number of proliferating cells (BrdU+) was counted in the subventricular zone (SVZ) and peri-infarct areas (n=9/group). a) Bar graph showing the absolute number of BrdU+ cells in SVZ and peri-ischaemic cortex. b) Low power (x100) photomicrograph from the peri-ischaemic tissue of vehicle treated or

platelet lysate (PLT) treated animals stained with an antibody against BrdU. Note that the number of BrdU+ cells is significantly increased in PLT treated animals compared to vehicle and FGF treated animals. HPF = high power field, Bar graphs 50 µm. Data are represented as mean ± SEM, * p<0.05.

b

a




Discussion

Our findings demonstrate that ICV injection of exogenous platelet lysate promotes angiogenesis, proliferation, survival and terminal differentiation in the cortex. These effects were associated with better functional outcomes and neuroprotection in treated ani-mals.

In addition to their well-known function in haemostasis, pla-telets also release substances that promote tissue repair, angiogen-esis and inflammation (19). Platelets are the first cells that adhere to sites of vascular lesions, where they secrete many growth fac-tors, chemokines and cytokines, and are capable of interacting with progenitor cells (20). Platelets release cytokines such as

VEGF, FGF (21, 22), PDGF, brain derived neurotrophic factor (BDNF) (23) and lipid mediators such as sphingosin-1-phosphate, which are all known to enhance angiogenesis and neo-vasculari-sation and promote neuronal survival and differentiation (24).

Platelets support mobilisation and chemotaxis of progenitor cells through release of chemokines such as stromal derived factor 1 (SDF-1), and also support survival, recruitment and differenti-ation of progenitor cells in vitro and in vivo (25, 26).

Membrane-bound signalling molecules, that can also be found on platelets (27), such as ephrin or PAR where also shown to play a role in survival, development, and plasticity of neurons (28) and may have been responsible for part of the effect seen in the current set of experiments.

Figure 5: Effects of platelet lysate on newborn cell differentiation. Terminal differentiation of proliferating cells was studied with cell-type spe-cific antibodies (n=9/group). a) Low power (x100) photomicrograph taken 90 days after stroke onset from the peri-lesion areas of vehicle treated animals (A) compared with platelet lysate (PLT, B). NeuN+/BrdU- cells are shown in the rectangular boxes, arrows point at double positive NeuN+/BrdU+ cells in the PLT treated animals. Nuclei were counterstained with DAPI (blue). Most of the newborn cells (BrdU+) in animals treated with PLT (65%) remained undifferentiated and expressed Nestin; the number of those neuronal stem/

progenitor cells was significantly larger at the infarct border zone compared to vehicle and FGF treated animals (b). 20% of newborn cells (BrdU+) in ani-mals treated with PLT differentiated to astrocytes and expressed GFAP; the number of those cells was significantly larger at the infarct border zone com-pared to vehicle and FGF treated animals (b). 15% of newborn cells (BrdU+) in animals treated with PLT differentiated to neurons and expressed NeuN; the number of those cells was significantly larger at the infarct border zone compared to vehicle and FGF treated animals (b). Bar graphs 50 µm in all pa-nels. Data are represented as mean ± SEM, * p<0.05.

a

b




Because of the multitude of trophic factors that are present in the platelets it is impossible to dissect out in in vivo studies such as the current one which of these factors affects recovery. However, our previous work (13) demonstrated that incubation with platelet microparticles (PMP) that contain similar mediators to those seen in the whole platelet albeit in smaller concentrations, leads to simi-larly increased eNSC proliferation and survival in-vitro and in-creased the differentiation potential of eNSC to glia and neurons. That study suggested that the effect of PMP was mediated by a number of these factors working in concert as the effects were re-duced but not abolished by the addition of specific blockers to FGF2, VEGF or PDGF to the medium whereas platelet factor 4 (PF4) inhibitors had no effect on eNSC (13). Hence, and because each of these factors activates specific receptors on eNSC and en-dothelial precursors, it is likely that they all contribute to the over-all observed effect. As shown in our previous work these effects were associated with increments in ERK and Akt phosphorylation, which are both implicated in cell survival, angiogenesis and prolif-eration (13).

It is important to note that at this stage it remains unclear whether or not proteins contained in PLT cross into the brain or just stimulate the eNSC within the SVZ.

In accordance with previous reports behavioural deficits began to improve after 14-20 days from stroke onset suggesting that be-havioural improvement was associated with an increase in neur-ogenesis (2, 3, 29). Proliferating cells accumulated in the cortex surrounding the injured tissue corroborating previous reports (3, 30). The location of newborn cells suggests that newborn cell mi-gration is influenced by the immediate neighboring tissue and is not entirely cell autonomous (31). These findings suggest that newborn cells migrate from the lateral ventricle SVZ towards the injured cortex (3, 30) and that platelets actively influence this mi-gration.

Furthermore, in accordance with prior studies we did not find a large number of newborn neurons at the immediate peri-infarct area. Rather, most of the BrdU+ cells at this area were immature, suggesting that terminal differentiation at this site if completed at all, may take a long time (3, 30). However, in areas slightly outside the immediate peri-infarct area we did observe an increase in the number of BrdU/NeuN double positive cells in agreement with previous studies (3).

Because our in vitro studies did not show a clear instructive pro-neuronal differentiation effect for PMP, the increased number of newborn neurons observed in vivo with PLT may be related to an absolutely higher number of surviving neural stem cells in the ischaemic milieu. This process may be aided by the well-known pro-angiogenic effects of platelets as NSC survive better in a specialised stem cell niche that combines young cells and young blood vessels (31-33). Therefore, our results also imply that PLT may aid in the formation of a secondary stem cell niche at the lesion border that may have an instructive role toward glial and neuronal differentiation following ischaemic stroke.

Our initial in vivo experiments used topical delivery of PMP to the brain surface (10). However, the clinical applicability of this delivery method in humans is complicated because of the need for

stereotaxic delivery into the brain. In the current set of experi-ments PLT, which are much easier to obtain, were delivered to ro-dent brains directly into the lateral cerebral immediately following PMCAO. This delivery mode is much more practical since it is constantly used in humans for cytotoxic drug delivery to the cer-ebral ventricle via an Ommaya chamber (34), which is a simple and safe bedside procedure that can be easily accomplished. Thus, this delivery method appears to be feasible, inexpensive and ad-vantageous compared with delivery of trophic factors delivered via mini-osmotic pumps to the ventricles or with the use of genetic manipulation using viral vectors to augment growth factor con-centrations in the brain. Nevertheless, it is clear that this delivery system is far from perfect and therefore, the current preliminary experiments should be viewed as a proof of concept to explore whether systemic or intra-thecal administration of platelets which will likely involve higher doses to ensure local concentration in the brain will also result in improved outcome.

In conclusion, we have shown that treatment with PLT can in-crease endogenous repair and improve outcome after brain injury. The application of platelets for treatment of stroke in humans may be feasible and should be further investigated to evaluate safety and efficacy in large animal models. Further study is also needed to identify the molecular mechanisms responsible for the increase in number and survival of neural cells that lead to improved func-tional outcome following brain injury.

AcknowledgementsWe thank Mr. Daniel Neiman for his help with the cell counting and Dr. Amalia Tabib. This work was supported by a grant from the Ministry of Science, Culture & Sport, Israel and by the Peritz and Chantal Sheinberg Cerebrovascular Research Fund, the Sol Irwin Juni Trust Fund. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Conflicts of interestNone declared.

What is known about this topic?• Platelets contain chemoattractants and mitogens that have a

major role in tissue repair.

• Platelets induce angiogenesis in vitro and in vivo.

• Platelets improve reperfusion in a rat myocardial infarction model.

What does this paper add?• Platelets increased endogenous neuronal stem cell (eNSC) prolif-

eration in a rat stroke model.

• Platelets induced angiogenesis and neurogenesis.

• Platelets enhanced neuroprotective effect.

• Platelets decreased behavioural deficits after brain ischaemia.




References1. Nakagomi T, Taguchi A, Fujimori Y, et al. Isolation and characterisation of neu-

ral stem/progenitor cells from post-stroke cerebral cortex in mice. Eur J Neuros-ci 2009; 29: 1842-1852.

2. Nakatomi H, Kuriu T, Okabe S, et al. Regeneration of hippocampal pyramidal neurons after ischaemic brain injury by recruitment of endogenous neural pro-genitors. Cell 2002; 110: 429-441.

3. Leker RR, Soldner F, Velasco I, et al. Long-lasting regeneration after ischaemia in the cerebral cortex. Stroke 2007; 38: 153-161.

4. Mazzucco L, Borzini P, Gope R. Platelet-derived factors involved in tissue re-pair-from signal to function. Transfus Med Rev 2010; 24: 218-234.

5. Nocito A, Georgiev P, Dahm F, et al. Platelets and platelet-derived serotonin promote tissue repair after normothermic hepatic ischaemia in mice. Hepatol-ogy 2007; 45: 369-376.

6. Rautou PE, Vion AC, Amabile N, et al. Microparticles, vascular function, and atherothrombosis. Circ Res 2011; 109: 593-606.

7. Arvidsson A, Collin T, Kirik D, et al. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med 2002; 8: 963-970.

8. Androutsellis-Theotokis A, Leker RR, Soldner F, et al. Notch signalling regulates stem cell numbers in vitro and in vivo. Nature 2006; 442: 823-826.

9. Parent JM, Vexler ZS, Gong C, et al. Rat forebrain neurogenesis and striatal neuron replacement after focal stroke. Ann Neurol 2002; 52: 802-813.

10. Hayon Y, Dashevsky O, Shai E, et al. Platelet Microparticles Induce Angiogen-esis and Neurogenesis after Cerebral Ischaemia. Curr Neurovasc Res 2012; 9: 185-192.

11. Brill A, Dashevsky O, Rivo J, et al. Platelet-derived microparticles induce angio-genesis and stimulate post-ischaemic revascularisation. Cardiovasc Res 2005; 67: 30-38.

12. Mause SF, Ritzel E, Liehn EA, et al. Platelet microparticles enhance the vasore-generative potential of angiogenic early outgrowth cells after vascular injury. Circulation 2010; 122: 495-506.

13. Hayon Y, Dashevsky O, Shai E, et al. Platelet Microparticles Promote Neural Stem Cell Proliferation, Survival and Differentiation. J Mol Neurosci 2012; 47: 659-665.

14. Mack M, Kleinschmidt A, Bruhl H, et al. Transfer of the chemokine receptor CCR5 between cells by membrane-derived microparticles: a mechanism for cel-lular human immunodeficiency virus 1 infection. Nat Med 2000; 6: 769-775.

15. Beni-Adani L, Gozes I, Cohen Y, et al. A peptide derived from activity-depend-ent neuroprotective protein (ADNP) ameliorates injury response in closed head injury in mice. J Pharmacol Exp Ther 2001; 296: 57-63.

16. Swanson RA, Morton MT, Tsao-Wu G, et al. A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 1990; 10: 290-293.

17. Shetty AK, Rao MS, Hattiangady B, et al. Hippocampal neurotrophin levels after injury: Relationship to the age of the hippocampus at the time of injury. J Neur-osci Res 2004; 78: 520-532.

18. Cheng K, Malliaras K, Shen D, et al. Intramyocardial injection of platelet gel promotes endogenous repair and augments cardiac function in rats with myo-cardial infarction. J Am Coll Cardiol 2012; 59: 256-264.

19. Rozman P, Bolta Z. Use of platelet growth factors in treating wounds and soft-tissue injuries. Acta Dermatovenerol Alp Panonica Adriat 2007; 16: 156-165.

20. Stellos K, Gnerlich S, Kraemer B, et al. Platelet interaction with progenitor cells: vascular regeneration or inquiry? Pharmacol Rep 2008; 60: 101-108.

21. de Boer HC, Verseyden C, Ulfman LH, et al. Fibrin and activated platelets coop-eratively guide stem cells to a vascular injury and promote differentiation to-wards an endothelial cell phenotype. Arterioscler Thromb Vasc Biol 2006; 26: 1653-1659.

22. English D, Garcia JG, Brindley DN. Platelet-released phospholipids link haemo-stasis and angiogenesis. Cardiovasc Res 2001; 49: 588-599.

23. Fujimura H, Altar CA, Chen R, et al. Brain-derived neurotrophic factor is stored in human platelets and released by agonist stimulation. Thromb Haemost 2002; 87: 728-734.

24. Ventimiglia R, Jones BE, Moller A. A quantitative method for morphometric analysis in neuronal cell culture: unbiased estimation of neuron area and number of branch points. J Neurosci Methods 1995; 57: 63-66.

25. Stellos K, Gawaz M. Platelet interaction with progenitor cells: potential impli-cations for regenerative medicine. Thromb Haemost 2007; 98: 922-929.

26. Schneider A, Kruger C, Steigleder T, et al. The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogen-esis. J Clin Invest 2005; 115: 2083-2098.

27. Prévost N, Woulfe DS, Jiang H, Stalker TJ, Marchese P, Ruggeri ZM, Brass LF. Eph kinases and ephrins support thrombus growth and stability by regulating integrin outside-in signalling in platelets. Proc Natl Acad Sci 2005; 102: 9820–9825.

28. Palmer A, Klein R. Multiple roles of ephrins in morphogenesis, neuronal net-working, and brain function. Genes Dev 2003; 17: 1429–1450.

29. Parent JM. Injury-induced neurogenesis in the adult mammalian brain. Neuros-cientist 2003; 9: 261-272.

30. Thored P, Arvidsson A, Cacci E, et al. Persistent production of neurons from adult brain stem cells during recovery after stroke. Stem Cells 2006; 24: 739-747.

31. Palmer TD, Willhoite AR, Gage FH. Vascular niche for adult hippocampal neur-ogenesis. J Comp Neurol 2000; 425: 479-494.

32. Ohab JJ, Fleming S, Blesch A, et al. A neurovascular niche for neurogenesis after stroke. J Neurosci 2006; 26: 13007-13016.

33. Nakagomi N, Nakagomi T, Kubo S, et al. Endothelial cells support survival, pro-liferation, and neuronal differentiation of transplanted adult ischaemia-induced neural stem/progenitor cells after cerebral infarction. Stem Cells 2209; 27: 2185-2195.

34. Blackshear PJ. Implantable drug-delivery systems. Sci Am 1979; 241: 66-73.


Documents

Platelet lysates stimulate angiogenesis, neurogenesis and neuroprotection after stroke