Transforming growth factor-ß1 and basic fibroblast growth factor in the injured CNS

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23 Wilson, C. and Cooper, S. M. (1989) Br. 1. Pharmncol. 98, 1303-1311

24 Quast, U. and Baumlin, Y. (1991) Elrr. /, PlrnrmacoL 200, 239-249

25 Yamagishi, T., Yanagisawa, T. and Taira, N. (1992) Nnutryiz Schmiedeberg’s Arch. Phnrmacol. 346, 691-700

26 Ito, S., Kajikuri, J., Itoh, T. and Kuriyama, H. (1991) Br. /. PharmncoL 104, 227-233

27 Itoh, T. et al. (1992) 1. Physiol. 451, 307-328

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30 Rasmussen, H., Kelley, G. and Douglas, J. S. (1990) Am. 1. Physiol. 258, L279-L288

31 Challiss, R. A. J., Patel, N. and Arch, J. R. S. (1992) Br. J. Pharmncol. 105, 997-1003

32 Schultz, J. E., Klumpp, S., Benz, R., Schiirhoff-Goeters, W. J. C. and Schmid, A. (1992) S&m 255, 600-603

33 Okada, Y., Yanagisawa, T. and Taira, N. (1992) Inp. 1. Pllnrnrncol. 60, 403.405

34 Okada, Y., Yanagisawa, T. and Taira, N. (1993) Nnun!y,z Scbmiedeberg’s Arch. PJnwmncol. 347, 438-444

35 3kada, Y., Yanagisawa, T. and Taira, N. (1992) E!lr. 1. Phnnr~nroi. 218, 259-264

36 Quast, LJ. and Baumlin, Y. (1988) Nazrnyn Sc/zmiedeberx’s Arch. Phnmmcol. 338, 319-326

37 Quast, U. et al. (1993) Mol. Pharmncol. 43,474-481

38 Edwards, G., Ibbotson, T. and Weston, A. H. (1993) /. Physiol. 467, 311

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40 Quast, U. (1991) Pfliigers Arch. 418

(Suppl.), R50 41 Foster, K. A., Arch, 1. R. S., Newson,

I’. N., Shaw. D. and Taylor, S. G. (1992) Ettr. 1. Pltnn~tacol. 222, 14%151

42 Greenwood, 1. A. and Weston, A. H. (1993) Br. 1. Phnrmncal. 109, 925-912

43 Choora. L. C.. Twort. C. H. C. and War& j. I’. T. (1992) Br. I. Plznnnacot. 105, 259-260

44 Wessler, 1. ef al. (1993) Nnn,r!/n Scbmiedeberg’~ Arch. Phnrmncol. 348, 14-20

45 Drieu la Rochelle, C. et al. (1992) J. Plznnuacof. Exp. ‘Jher. 263, 1091-1096

E4080: (E)-N-[(3-((N’-[2-(3’,5’- dimethoxyphenyl)ethyI)-N’-methyl) - amino)propyl]-4-(4-(lH-imidazol-1-yl) phenyl-3-butenamide Ki4032: N-(2-acetoxyethyl)-N’-cyano-3- pyridine carboximidamide

ransform

factor in the injure Ann Logan and Martin Berry

After injuries that penetrate the mature brain or spinal cord, damaged axons initially show a growth response, but later their regeneration is aborted as LI dense permanent scar is laid down within the core of the wound. Functional recovery from such injuries is poor and morbidity is severe, particularly for those patients with spinal cord damage. Clinically, no long term therapeutic treatments have beeu developed that might inhibit scarring and promote neuronal growth. Consequently, the prevalence of patients permanently disabled from head and spinal cord injury is high, estimated nt more than 1:lOOO of the population of North America (Off ice of Technology Assessment USA, 1990). Ann Logan and Martin Berry define the mechanisms that underlie the wound healing response in the CNS and discuss the rationale for the development of novel therapeutic strategies.

Penetrating injuries of the CNS initiate a complex cellular wounding response’ (summarized in Fig. 1). Immediately after trauma, excitatory amino acids are released, and their excitotoxicity ultimately leads to neuronal cell death2. The wound becomes filled with a plug of haematogenously derived material, including monocytes, which migrate from the blood into the damaged nervous tissue where they transform into macrophages. This extravasation may be mediated by the local sequential expression of selectin and integrin

A. Logan is Lectairer in the Department of Clinical Chemistry, University of Birmingham, Edgbaston, Eirmiitgham, UK 815 2TH artd M. Berry is Professor and Chairmnn in the Division of Amztomy and Cell Biology, UMDS, Guy’s Campus, Londorr, UK SE1 9RT.

cell adhesion molecules by endothelial cells in the microvascula- ture3, a process activated by molecules released from the blood clot. A wave of reactive gliosis is then initiated in the surrounding nervous tissue, which spreads from the borders of the wound by the proliferation and migration of astroglia and probably also microglia. It is still conjectural whether a population of microglia in the wound are derived from invading monocytes4. In subsequent days, injury-responsive microglia exhibit neurophagy within the wound margins, acting not just as phagocytes of necrotic tissue, but also recognizing axotomized neurones and destroying them by the release of proteases5. The clearance of necrotic tissue is

aided by macrophages that are recruited to the site of injury. Activated astrocytes reorganize a limiting membrane, which becomes continuous with the glia limitans extema, and eventually delineates the cut edges of neuropil. Fibroblasts are chemoattracted into the core of the wound from the meninges, initiating matrix deposition and the formation of a basal lamina which underlies the glial membrane. Neovasculariz- ation occurs in the surrounding tissue. Comprcmised axons that have survived the initial injury, subsequently produce growth cones and form synapses. Yet, after a few days of growth, regeneration is aborted and the newly formed sprouts degenerate, poss- ibly as a consequence of excitotoxic damage and microglial targeting. This, together with the concomitant development of a double glia limitans in the wound, makes functional reconnection of severed neural pathways im- possible.

Is healing helpful? The normal injury response

leads to rapid wound closure, reformation of the blood-brain and blood-cerebrospinal fluid barriers, and an appropriately controlled inflammatory response. However, it is often argued that some aspects of this response mitigate functional recovery of the tissue. The transient nature of the regenerative response and the formation of a fibrous scar are clearly not helpful to the reconnection of traumatized neural pathways. Why such a

dpl

- Haematogenous plus Invasion by fibroblasls and macrophatws

W

Collagen scttr condenses Mature glial /collagen scar

Fig. 1. Spatial and tempera/ cellular changes occurring after a cortical injury lo one cerebral hemisphere of an adult rat. dpl, days post lesion.

functionally deleterious response to CNS injury has evolved is para- doxical. There may be a selective advantage for rapid wound closure to ensure both a minimal inflammatory response and reconstitution of the extracellular environment in which neurones can re-establish function. Also, the fundamental difficulty of accurately reconstructing the complex circuitry of the CNS after damage may mean that no reconnection is better than bad reconnection.

Orchestraters of the wounding response

Multiple haemstogenous and tissue-specific growth factors and cytokines are locally released after damage, and interact to control the cellular changes just described’. Some of these trophic events are now beginning to be understood.

One of the first events is the de- livery into the wound of platelet- derived growth factor (PDGF) and transforming growth factor-@ (TGF-j3s) by platelet lysis. This is accompanied by the invasion of cells of the monocyte/macrophage lineage, which are the most per- sistent cell types associated with the wound. These cells then produce numerous cytokines, such as tumour necrosis factors, inter- leukins, TGF-l3s, and fibroblast growth factors (FGFs), all of which may themselves be trophic and may also stimulate the production of trophic substances from target cells7-9. Reactive astrocytes are thought to be a primary source of multiple growth factors and cytokines after injury, including basic FGF and TGF-~s~,~. Damaged neurones release a number of trophins into the wound site, including the TGF-Bs, and acidic

TiPS - September 2993 Wol. 141

and basic FGF%r’. Finally, the cerebrospinal fluid may be a source of active molecules, such as insulin-like growth factors, which may influence neuronal and glial responses”. The orchestration of this complex set of trophic factors must be exact in order to initiate tissue-specific responses. Defi- nition of the relative contribution of individual factors in nivo is difficult, yet studies are now estab- lishing TGF& and basic FGF as major regulators of the injury response.

Five distinct isoforms of TGF-fis have been described, although only TGF-j3i, -pa and $3 are found in mammals (see Ref. 12 for review). Each isoform is encoded by a separate gene, and each has a distinct 5’-regulatory sequence. The peptides are all secreted from cells in a biologically inert, latent form, made up of a mature TGF-l3 dimer together with a latency- associated protein. Before the TGF-j3s can bind to their receptor(s), they must first be activated by dissociation from this complex.

The TGF-Ps are trophic regulators of cell proliferation, differentiation and differentiated function, mediating multiple aspects of mesenchymal and epi- thelial cell activities in many tissues and at all developmental stages. In culture, the bioactivities of the TGF-l3 isoforms are often interchangeable, but their speci- ficity of action in many tissue systems in viva is noted. The involvement of TGF-fis in wounding responses is well established in peripheral tissues, where they are potent desmoplastic agents and inflammatory mediators. TGF-Pi is released from platelets and secreted from cells of the monocytel macrophage lineage at the earliest stages of the injury re- sponseP*‘3. Within wounds, the cytokine has a broad spectrum of influences; it acts not only as a mediator of inflammatory and angiogenic responses, but also as an organizer of extracellular matrix deposition, both by affect- ing fibroblast expression of col- lagens, fibronectins and elastin, and by modulating the activity of matrix proteinases and protease inhibitors, thereby remodelling tissues after damage (reviewed in Ref. 14). In addition, it is

TiDS - September 1993 [Vol. 141

suggested that TGF-PI controls cell migration and differentiation by influencing the expression of cell adhesion molecules within responsive tissues15. Numerous studies have illustrated the potential use of exogenous TGF-fls to enhance the repair of bone and skin16*17. Conversely, it is reported that neutralizing antibodies to TGF-fil can reduce fibrous scar formation in skin, with potential cosmetic effects17.

At first sight, TGF& is not a likely CNS trophic factor. In the mature animal, its mRNA is not normally expressed at significant levels in neural tissues, although the peptide has been identified in the meninges and choroid plexu&‘*. However, new evidence is accumulating to suggest that TGF-P1 may play a critical role in the CNS, particularly under selected pathological conditions.

Studies have shown that astrocytes and neurones, which are effectively ‘wounded’ by their removal from tissues into mono- layer culture, express TGF-PI mRNA and peptide in vitro”. The cytokine is known to stimulate the proliferation of astrocytes in primary culture”. The peptide also affects astrocyte migration and interaction”, and the expression of plasminogen activator, fibronectin, collagen, laminin”, glial fibrillary acidic protein (an intermediate filament protein)22, interferon-induced major histo- compatibility complex class-II antigen23 and cell adhesion molecules such as Ll and N-CAM24. The effects of TGF+ on cultured neurones has been less exten- sively studied, but there is some evidence that the peptide can promote neuronal survival and neurite outgrowth by modulating the effects of neurotrophic factors such as basic FGF and NGF25.

More direct evidence of a role for TGF& in CNS pathologies has come from in viva experiments. Immunoreactive TGF-6~ is seen in macrophages and astrocytes within neural tissue infected with HIV-l (Ref. 26). After mech- anical lesioning8,27,28, and hypoxic ischaemia29, TGF-PI is seen to be rapidly and transiently expressed within damaged neural tissue. AS

in the periphery, the cytokine is released from platelets and is also translocated into the wound by invading cells of the monocyte/

Fig. 2. Dark field micrographs show- ing transforming growth factor-& (TGF-p,) mRNA expression in neural tissue two days (2d) afler an injury to the cerebral cortex of an adult rat The upper panel shows a section through the wound site probed with an antisense (T7) cRNA probe specific for TGF-/J, mRNA. The lower panel shows an adjacent section probed with a sense (T3) control probe. The curved open arrows show the margin of the lesion. the straight open arrows show the meninges.

macrophage lineage. Within hours of injury, TGF-(3, is also expressed locally by compromised neurones and glia, and distally at multiple sites including the meninges, choroid plexus and the endothelial cells of the micro- vasculature (see Fig. 2).

Within damaged CNS tissue, TGF-P1 seems to have multiple influences. Some of these have been demonstrated directly by infusing specific anti-TGF-fi, antibodies into CNS wounds to neutralize endogenous TGF-PI

339

!Ref. 30). The consequences of this treatment include a reduction in the number of macrophages and fibroblasts invading the wound, and a dramatic inhibition of matrix deposition. It is note- worthy that, although a normal reactive gliosis response is observed, the activated astrocytes neither migrate nor associate to form a limiting membrane bordering the wound (see Fig. 3). The suppression of mesodermal scar formation after treatment with anti-TGF-6, antibodies demon- strates the primary importance of this cytokine for the initiation of CNS scarring. Since it is c!ear that scar formation in this instance limits neuronal regeneration, the therapeutic potential of locally applied TGF-PI antagonists for patients with CNS injuries is implicit.

Notwithstanding the implied clinical importance of scar reduction in the CNS, the studies with TGF-PI antagonists have provided no evidence for enhanced regeneration in scar-inhibited wounds. It seems that the physical barrier of scar material is not the sole factor limiting neuronal growth after injury. Additional strategies are required in order to reduce neurotoxicity within wounds, and to promote the regenerative response of damaged neurones. Identification of key endogenous neurotrophic factors will be central to this therapeutic development.

Basic FGP Basic FGF (also referred to as

FGF-2) is a member of a family of homologous proteins that are potent effecters of a wide range of cell types (for review see Ref. 31). Most recently, a ninth member of this family has been identified3’. The potential of basic FGF as a neurotrophic factor has long been recognized. The peptide and its high-affinity receptors are expressed within CNS tissue. In the normal adult brain, basic FGF is present in neurones and glia, in the vascular basement membrane, the meninges and in the epen- dymal cells lining the ventricular system. Glia and neurones syn- thesize both the growth facto?3 and its receptor(s)34 throughout the brain and spinal cord; particularly high levels of expression of basic FGF are seen in discrete

340

Fig. 3. Effects of injections of neotral- riing anti-TGF$ IgG. fhotomicro- graphs show sections taken through the site of a stereotactica//y defined cerebral lesion in adult rats fourteen days after daity injections of either a: control tgG or b: anti-TGF-@, IgG. Sections were floorescently-stained for fibronectin and show a dramatic reduction in mafrix material in the anti-TGF-fl, IgG injected animals.

populations of neurones, such as the induseum griseum, fasciola cinereum, field CA2 of the hippo- campus, the septohippocampal nucleus, cingulate cortex and the subfomical organ. The functional significance of this discrete pattern of expression is not established.

Basic FGF is also found throughout the developing CNS35, where it may be important for the maturation of both acetylcholine- and dopamine-containing neurones. Basic FGF promotes the survival in culture of a wide range of foetal CNS neuronal populations, including cortical, hippocampal, striatal, septal, hypo- thalamic, mesencephalic and ciliary ganglionic neurones31. It enhances outgrowth of their neur- ites, and promotes choline acetyl- transferase activity in septal cultures36 and dopamine uptake in mesencephalic cultures37. In suprort of a neurotrophic role for this peptide in the developing CNS, exogenous basic FGF is reported to rescue neurones in viva from programmed cell death3s ultd also from the photoreceptor degeneration associated with an inherited retinal dystrophy39.

Despite the early identification of basic FGF in the normal CNS, little is known of its physiological function. Studies in vivo do provide evidence, albeit circum- stantial, for a neurotrophic role for endogenous basic FGF after injury. For example, a number of reports have demonstrated in- creased immunoreactive and bio- active basic FGF in the chemi- cally40 and mechanically lesioned brain9,4’,42, with enhanced expression localized to neurones, glia and vascular endothelial cells within the damaged neuropil (Fig. 4). Similarly, transient forebrain ischaemia is reported to induce basic FGF expression in neurones and astrocytes43. Interestingly, key experiments have not been described that might establish a role for basic FGF in the CNS, such as the consequences for neuronal function of endogenous neutralization of basic FGF activity in normal or damaged CNS; they may reflect the intracellular, and therefore functional, sequestration of the protein in this tissue. The development of noncompetitive antagonists acting at the FGF receptor44 and antisense DNA strategies45 may aid direct testing of the physiological functions of this peptide.

Other in vivo experiments have provided indirect evidence of a neurotrophic role of basic FGF in damaged CNS tissue, and indicate its potential as a therapeutic agent to ameliorate neurodegenerative conditions (reviewed in Ref. 6). When given exogenously, the protein promotes the survival of central neurones after chemical insult and axotomy. It also atten- uates the downregulation of hippocampal choline acetyltrans- ferase activity induced by partial fimbria transection. In addition, recent data have shown that basic FGF causes a reduction in hippocampal neuronal death after glutamate-induced axotomy, by raising the threshold for glutamate neurotoxicity. This obser- vation suggests that basic FGF may play a protective role against excitotoxic neural damage in the mature CNS.

Basic FGF in vitro has a broad range of activities on multiple cell types31, and its potential influences in the CNS in viva may thus extend beyond direct neuro- tropism. Basic FGF is mitogenic

TiPS - September 2993 lV01.141

for oligodendrocytes and astrocytes. It stimulates the migration and the functional differentiation of astrocytes, whereby plasminogen activators are released and intermediate filament protein, glial fibrillary acidic protein, glutamine synthetase and SlOO protein are all expressed. Further- more, it modifies the morphological maturation of astrocytes, by re- arranging intermediate filaments, lengthening cellular processes and changing astrocyte membrane structure. Basic FGF is one of the most potent angiogenic factors so far identified, being both mito- genie and chemotactic for endothelial cells. For example, intra- venticular46 and parenchyma147 administration is reported to promote cerebral angiogenesis in damaged neural tissue Jter chronic forebrain ischaemia. As its name suggests, basic FGF is also a mitogen and chemoattractant for fibroblasts, promoting the formation of granulation tissue within peripheral wounds in vivo4’. Its involvement in scar formation in the CNS remains to be demonstrated, but is probably minor when compared to the other potent fibrotic agents, such as PDGF and TGF&, which are also present within wounds.

It seems, therefore, that basic FGF may be involved in multiple aspects of the CNS wounding response, including neuronal sprouting, reactive gliosis, angiogenesis and fibrous scar formation. Whilst some of these responses are beneficial to neuronal survival and regeneration, they are potentially balanced by functionally deleterious influences. Until a precise role is defined in vivo, it is difficult to predict the therapeutic value of basic FGF as a neurotrophic factor for neurodegenerative conditions.

Manipulation of the wounding response

There is now considerable evidence that both basic FGF and TGF-P1 are important trophic regulators of the injury response of the CNS, and that manipulation of their activities in damaged tissue can influence the wounding response beneficially. However, it is also clear that a multiplicity of other trophic and tropic (mechanochemical) factors determine the final regenerative response of

TiPS - September 1993 [Vol. 241

damaged CNS neurones. Neuro- trophic factors, proteolytic en- zymes, glial inhibitory factors, the mechanochemical substrata and scar deposition are all cited as examples of elements that interact to determine the axonal response. Since it is established that adult central neurones can re- grow if provided with a com- patible trophic and mechanochemical environment4’, it would seem reasonable to envisage a strategy of trophic and tropic manipulation of CNS wounds to establish a neuronally supportive environment.

Regardless of the neurotrophic strategy devised, the scar material that is deposited within wounds is a major obstacle for neuronal regeneratiorP, and must be over- come before functional reconnection is possible. The studies demonstrating that a locally applied neutralizing TGF& anti- body can prevent fibrous scarring within CNS wounds may resolve this problem30. The identification of a dermatan sulphate proteo- glycan, decorin, which acts as a pan-TGF-P antagonist51, provides another reagent with a similar activity but with the therapeutic advantages of being smaller, more mobile, less labile and less im- munogenic. In vivo, this molecule has already been shown to sup- press the accumulation of pathological matrix material in kidney glomeruli in experimenta) glom- erulonephritis” and in neural tissue after penetrating CNS injuries (Logan, A. and Berry, M. unpublished observations). The availability of soluble TGF-P receptor may provide another molecule capable of antagonizing endogenous TGF-P activity within wounds. However, even if scarring is reduced, it seems that damaged CNS axons are still in- capable of regeneratior?O. Thus, the development of a co-ordinated neuronotrophic strategy is also required.

For many severed adult CNS axons, the environs of a peripheral nerve are conducive to regrowth, providing a suitable substratum for axon al extension53. For example, regeneration of some 10-15% of retinal ganglion cell axons in the adult optic nerve is promoted by suturing a peripheral nerve to the cut end of the optic nerve, Retinal ganglion cell

Fig. 4. lmmunoreactive basic fibrobasf growth factor (FGF) in the neural tissue bordering a 5 day-o/d wound in the raf cerebral hemisphere. Basic FGF is seen localized to damaged neuronws (n), astrucytes (a), and endorhelial ce!/s of the microvasculalure (e). The scale bar is 5pm.

axons traverse the anastomosis, and grow for long distances inside the Schwann cell basal lamina tubes within the grafts54. If all Schwann cells are destroyed in the peripheral nerve by freeze- thawing before grafting, no retina) ganglion cell axons regenerate into the implant. This is presumably because the appropriate neurotrophic substances are absent, since permissive substrates for growth (the basal lamina tubes) persist within the graft55*56. Thus, the continued supply of appropriate neurotrophic factors would seem crucial for a sustained growth response of axotomizcd neurones.

On the basis of these studies, we predict that, within the CNS, a neuronotrophic strategy that combines inhibition of endogenous proteases with provision of exogenous neurotrophic factors and anti-inflammatory substances, may prove important for limiting neurona) death and facilitating axonal growth over a suitable growth substratum. Indeed, Than09 has effectively used protease inhibitors irl uivo to promote survival and axon growth of axotomized retinal ganglion cells. In addition, it has recently been demonstrated that inter- leukin-1 receptor antagonistss7

342 TiPS - September 1993 [Vol. 141

and lipocortin-I (an endogenous phospholipid)’ can significantly reduce the neuronal degeneration associated with the excitotoxicity resulting from forebrain ischaemia. Both are thus potential neuroprot~tive agents for damaged CNS tissue.

Although inhibition of axon growth in the adult CNS by oligodendrocytes and/or components of central myelin may mediate the failed regenerative response in the CNS”, otherss9 have shown that axons do not regenerate in the CNS even when these two elements are absent. molecules derived from astrocytes also may inhibit axonal regrowthbO, suggesting that neutralization of these inhibitory factors may prove an additionai strategy to promote neuronal regeneration.

Certainly, the limited ava~labili~ of endogenous neurotrophic factors in the injured adult CNS contributes to the observed un- sustained axonal growth response. As previously described, exogenously supplied neurotrophic factors (such as basic FGF) can promote nerve regrowth and survival in vim However, until there is a better understanding of the rofe that endogenous neurotrophic factors and their receptors play for diverse groups of CNS neurones, it will be difficult to set forth a comprehensive neuronotrophic strategy to modulate wound healing in the CNS. Factors such as nerve growth factor, acidic and basic FGF, ciliary neurotrophic factor, brain derived growth factor, neurotrophin3, midkine and pleiotrophin need systematic evaluation. Identification of the endogenous trophic factors and their receptors that operate within damaged CNS tissue may impli- cate physiologically relevant molecules with clinical potential. The problem of providing appropriate targeting cues for regenerating axons is beyond the scope of this review, but must also be addressed if we are to develop a co-ordinated clinical strategy for the reconnection of neural pathways.

0 q q

One of the key chdenges for clinical neurological research is the development of effective treatments for patients with compromised CNS pathways to promote functional recormection

of their severed neural tracts. Because the wounding response is complex and multifacto~al, a thorough understanding of its biological regulation is an important first step towards this goal. Progress is rapidly being made in the identification of key molecules regulating the injury response, and it is now clear that mod~ation of their activities within damaged CNS tissue can alter the course of wound healing. Basic FGF and TGF-PI may prove to be clinically significant molecules in this regard. However, it seems that changing the CNS environment into one conducive for sustained regeneration, and thereby facilitating functional reconnection of damaged neural pathways, will ultimately involve manipulation of multiple trophic and tropic components. The local application of cocktails of specific peptide agonists and antagonists to CNS wounds in order to achieve trophic transfo~ation of lesion sites may prove to be an entirely appropriate therapeutic strategy.

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50 Reier, P. J.. Stensaas, L. J. and Guth, L. (1983) in Spinal’ Cord Reconstruction (Koa, C. C. and Reier, P. J., eds), pp. 163-195, Raven Press

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J. ‘3. and Silver, J. (1991) J. Neurosri. 11, 3398-3411

New editions Pharmaceutical Dosag? Forms: Parenteral Medications, Volume 3. Second edition

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Parasitology Today 100th Issue

P~rffs~#o~~g~ T~~~~, a sister journal of TiPS, is celebrating its 100th issue in October by offering a 20% discount on the 1993 subscription price. Sub- scribe to ~~r~s~~o~ogy ~~~~~ during the month of October, using the order form in that issue, and you will receive 12 monthly issues for only fi2/$78.

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Documents

Transforming growth factor-ß1 and basic fibroblast growth factor in the injured CNS