Storage, metabolism, and processing of 125I-fibroblast growth factor-2 after intracerebral injection

BRAIN RESEARCH

ELSEVIER Brain Research 665 (1994) 285-292

Research report

Storage, metabolism, and processing of 125I-fibroblast growth factor-2 after intracerebral injection

Ana Maria Gonzalez a.* '+, Laurie S. Carman u, Michael Ong a, Jasodhara Ray b, Fred H. Gage b, Clifford W. Shults b,c, Andrew Baird a,+

a Department of Molecular and Cellular Growth Biology, The Whittier Institute for Diabetes and Endocrinology, 9894 Genesee Avenue, La Jolla, CA 92037, USA

b Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA c Neurology Service, VA Medical Center, San Diego, CA 92093, USA

Accepted 6 September 1994

Abstract

Basic fibroblast growth factor (FGF-2) is a potent trophic agent for both neuronal and non-neuronal cells of the mammalian CNS. It can enhance survival and neurite outgrowth of a variety of neuronal types in vitro and in vivo, and recently has been shown to stimulate neuroblast proliferation in culture. To determine the most effective means of introducing FGF-2 into the brain, and to further our understanding of the behavior of exogenous FGF-2 following intracerebrai injection, we examined the diffusion and degradation of 12SI-FGF-2 following intraventricular or intraparenchymal injection. SDS-PAGE and autoradiography show that when radiolabelled FGF-2 is injected into the parenchyma of the rat brain, it remains at the site of injection where it is detectable for several days. During this time, it is slowly metabolized to 2 specific heparin-binding metabolic fragments that are virtually identical to the ones described for its metabolism by neurons and astrocytes in vitro. Microscopic examination and autoradiography of these tissue sections show that within these areas, FGF-2 diffuses throughout the site of injection. Initially, it migrates along adjacent fiber tracts, binds to specific cells and to basement membranes of the microvasculature, but later on it remains associated to basement membranes and non-neuronal cells. Based on its slow clearance and slow rate metabolic degradation, this FGF-2 is presumed to be in a sequestered form and to have limited activity. In contrast, the intraventricular injection of 125I-FGF leads to a rapid clearance, with some binding to ependymal cells lining the ventricles and little translocation into the parenchyma. Based on these studies, we conclude that the distribution, half life, and activities of FGF-2 can be dramatically affected by the mode selected for its administration. This may derive from its high affinity for immobilized heparan sulfate related proteoglycans. Accordingly, in the design of strategies to test the neurotropbic potential of FGF-2, the ideal delivery protocol will require more than a slow release of locally acting growth factor and also a mechanism that retains the molecule at the desired site of action, to avoid sequestration in compartments adjacent to or distal from target cells.

Keywords." FGF-2; Neurotrophic factor; Injury repair; Angiogenesis

I. Introduct ion

Basic fibroblast growth factor (FGF-2) is a pluripo- tent growth factor that appears to play a variety of roles during development in normal tissue homeostasis and in injury repair. Al though it is widely distributed in

* Corresponding author. Fax: (1) (619) 457 5534 at "Present address" (see:following footnote).

+ Present address: The Scripps Research Institute, Department of Cell Biology, 10666 North Torrey Pines Rd., La Jolla, CA 92037, USA.

0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 0 9 7 - 8

both developing [21] and adult mammals [4], its m R N A is virtually undetec table in peripheral tissues and the protein often localizes to extracellular matrix [18,21, 26,47,52]. In contrast, its m R N A in the brain is readily detectable [11,41] and the protein localizes intracellu- larly in neurons and glia.

While in vitro and in vivo studies indicate that FGF-2 has potent t rophic effects on injured neurons, its physiological funct ion remains unknown. In vitro studies have shown that FGF-2 promotes neuri te outgrowth of a variety of neuronal types [15,27,46,48,49]. Indeed, following CNS injury, FGF-2 m R N A and protein increase significantly in the injured CNS [7,16,19,

286 A.M. Gonzalez et al. / Brain Research 665 (1994) 285-292

28,30,32,34]. Several groups have demonstrated that local infusion of FGF-2 into a lesion site promotes the survival of damaged nerve cells [1,2,35-37,43], and it may contribute to astroglial [14,20,40] and endothelial cell [32,39,45] proliferation. In our hands, however, the neurotrophic effects of FGF-2 in vivo have been, at best, variable (unpublished). We thus reasoned that a better understanding of what happens to exogenously- injected FGF-2 in vivo might help determine the most effective means for introducing it into the injured brain. In the experiments reported here, we used 1251- FGF-2 to examine the diffusion, binding, and degradation of FGF-2 following intracerebral (parenchymal) or intraventricular injection. The results suggest that while the growth factor remains at the site of injury, its association with basement membranes may limit its bioavailability. If so, these findings could explain the variable responses seen to FGF administration in the injured CNS.

2. Materials and methods

Coronal brain frozen sections (40 p.m), corresponding to the regions also selected for SDS-PAGE analysis, were dipped in Kodak NTB-2 emulsion and exposed for 2 weeks at 4°C. Sections were developed with Kodak D-19 developer for 3.5 min at 17°C, rinsed in water, and fixed for 2 min. Slides were rinsed for 45 min and counterstained with Cresyl violet for Nissl substance.

2.4. Regional dissection and distribution analyses of the brain

Tissues corresponding to frontal cortex, striatum (site of injection), hippocampus, rostral midbrain, and cerebellum were collected by dissection under a stereomicroscope according to Schults et al. [42]. The radioactivity in dissected tissues was counted on a manual gamma counter. SDS-PAGE and autoradiography were performed on extracts of the tissues obtained from the injection site. In these studies, the tissues were homogenized in extraction buffer [17] con- taining enzyme inhibitors and centrifuged. An aliquot of heparin- Sepharose (100 /xl) was added to the supernatant and incubated overnight at 4°C. The heparin-Sepharose-associated material was washed by centrifugation with PBS and 0.6 M NaCI in 10 mM Tris buffer. Fifty microliters from each sample were counted in a manual gamma counter and 800 cpm per tissue was solubilized with Laemmli's sample buffer and electrophoresed by SDS-PAGE using 15% polyacrylamide, 0.1% SDS. The gel was dried, laid on X-ray film and exposed for 2 days, 1 week and 2 weeks for optimal development.

2.1. Preparation of 125I-FGF-2 and 1251-NGF

Recombinant human FGF-2 [8] and NGF (Syntex, CA) were iodinated by the lactoperoxidase method, lzSI-FGF-2 was then puri- fied by heparin-Sepharose affinity chromatography as previously described [6]. The specific activity averaged 105,000 cpm/ng.

2.2. Animal surgery and injections

All animal procedures were carried out with strict adherence to the guidelines set out in the NIH Guide for the Care and Use of Laboratory Animals, National Institutes of Health Publications No. 80-23. Thirty male Sprague-Dawley rats (250-300 g) received intrastriatal injections of 125I-FGF-2 (105 cpm/25/z l ) using the stereotaxic coordinates (measured from Bregma) of 0.2 mm anterior, 3.5 mm lateral, and 4.5 mm ventral to dura. Injections were made over 5 min, and the needle was left in place an additional 4 min to prevent reflux. At various times after injection (15 min, 4 h, 2 days, 4 days, and 7 days), 2 rats from each group were decapitated, their brains were dissected into 14 regions (left and right - rostral neocortex, striatum (injection site), caudal neocortex, hippocampus, midbrain, cerebellum, and anterior and posterior pituitary)] and tissue was frozen in liquid nitrogen. The remaining 4 animals from each time point were processed for autoradiography (see below). A second set of 6 rats received intraventricular injections of 25 ~1 of 125I-FGF-2 and 2 animals were sacrificed at 2, 4, and 7 days for autoradiographic studies. Stereotaxic coordinates for intraventricular injections were 0.7 mm anterior, 1.2 mm lateral, and 3.5 mm ventral to dura. Injections were made over 5 min, and the needle was left in place for an additional 4 min to prevent reflux. 125I-NGF and physiological saline were used for control studies.

2.3. Perfusions and autoradiography

Animals were heavily sedated with chloral hydrate and perfused through the heart with 150 ml of saline followed by 300-400 ml of 4% paraformaldehyde in phosphate buffer solution (PBS). Brains that were collected for autoradiography were left in this fixative overnight, cryoprotected in 20% sucrose-PBS and stored at -20°C.

3. Results

3.1. Regional distribution of 125I-FGF-2 in the brain

Because several investigators have suggested that FGF-2 is transported anterogradely and retrogradely in

100

7 5 - - - - ' g - - - Frontal Cortex

--~ ~ Striatum (Injection Site 3 ~ Hippocampus ~ Rostral midbrain

~" 5 0 - ~ ~

"7.

2 5 -

0 24 48 7 96 120 144 168

Time after Injection (Hrs)

Fig. 1. Regional distribution of 1251-FGF-2. The radiolabeled counts in dissected tissue show no distinct pattern of accumulation within any brain region, and the majority of activity is always at the injection site. By 4 h after injection, 58% of injected activity remained within the injection site, and by 2 days this had decreased to 28%. By 4 and 7 days this activity is further decreased to 19% and 4%, respectively.

A.M. Gonzalez et al. / Brain Research 665 (1994) 285-292 287

vivo [5,11,23,24], we first quantified the amounts of radiolabeled FGF-2 after a regional dissection of the injected brains (Fig. 1). The results show that the majority of radioactivity remains in and around the injection site. After 4 h, 58% of injected activity remains within this area, and 48 h later 28% of the original radioactivity is still present. By 4 and 7 days after the injection of FGF-2, the remaining label is decreased to 19% and 4%, respectively. At all of these time points, little radioactivity is detected in the frontal cortex, the hippocampus, the rostral midbrain, cerebellum or pituitary. It would thus seem that the radioiodi- nated growth factor remains locally present at the site of injection. We thus determined whether it remains intact or is metabolized in situ.

~ cn ~n

t ~

Fig. 2. Metabolism of 1251-FGF-2. SDS-PAGE analysis shows after 4 hours the FGF-2 present in brain tissue corresponds to the 18-kDa molecular form. By 2 days after injection, in addition to the 18-kDa band, 2 bands of lower molecular weight can be detected. Four days after injection the 18-kDa band remained, but by 7 days the 18-kDa band is barely detectable. This suggests that the silver grains remaining in tissue in the 4- and 7-day autoradiographs probably reflected localization of FGF-2 metabolized fragments. SDS-PAGE is performed on extracts of the tissues obtained from the injection site.

3.2. Metabolism of 125I-FGF-2 in the brain

Numerous studies have described a specific metabolism of FGF-2 that occurs [9,33,50] after its internalization into target cells. This metabolism gener- ates specific, long-lived metabolic fragments of the molecule. We thus reasoned that, if the injected FGF-2 is rapidly utilized in vivo, it should be degraded imme- diately and these long lived radiolabeled fragments should be detectable by SDS-PAGE analysis and autoradiography. Surprisingly, after 4 h, the t25I-FGF-2 injected remains in its original 18-kDa form (Fig. 2). It is only 2 days later that the 2 bands of lower molecular weight are detected in the tissue extracts. By 4 days,

¢2 D

Fig. 3. Distribution of radiolabelled FGF-2 in vivo. Dark field micrographs showing the distribution of FGF-2 after different times of injection. Four hours after injection, radioactivity is very densely localized within the injection site and along the tract of fibers forming the corpus callosum (Panel A). By 2 days after injection, the pattern of radioactivity is very similar to that seen at earlier time points, but there is a gradual decrease in density of silver grains over the parenchyma and an increase over blood vessels (Panel B). Four days after injection, the radioactivity is still localized in the corpus callosum and at the site of injection, and the vessels surrounding the scar are highly labeled with silver grains (Panel C). This pattern remained 7 days after injection in the striatal parenchyma, where most of the radioactivity is concentrated over the vessels in the area of the injection (Panel D).


S D S - P A G E and autoradiography reveal that the original 18-kDa form of F G F - 2 is significantly degraded. By 7 days no ~25I-FGF-2 is detectable. This long t ime course suggests that while the FGF-2 is re ta ined at the site of injection, it is relatively protected from degradation, presumably by its association with cell surface and extracellular proteoglycans [3]. In order to address this possibility, we analyzed tissue sections by autoradiography.

3.3. Distribution o f radiolabeled FGF-2 in the brain

We a t tempted to identify the structures to which 125I-FGF remains associated at the inject ion site in order to explain its relatively long half life and appar-

ent stability in vivo. Four hours after an inject ion of F G F - 2 into the str iatum, the radioactivity is densely localized to the area of inject ion and diffuses along the tract of fibers forming the corpus callosum (Fig. 3A).

A

Fig. 4. Specific localization of exogenous FGF-2 in the area of injection. The ependymal cells lining the lateral ventricle, ipsilateral to the injection, show a high intracellular accumulation of clusters even 4 hours after injection (Panel A). These clusters are still present 7 days after injection, although in lower amounts. However 4 days after intraventricular injection, no intracellular clustering over ependymal cells is seen, although some silver grains are localized in the apical and basal side of ependymal cells (Panel B). Two days after injection, there is a dense accumulation of silver grains over blood vessels, which is more evident after 7 days (Panels C and D). Four hours after intrastriatal injection, neurons from the substantia nigra compacta, ipsilateral to the injection, contain small amounts of radiolabeled FGF-2 (Panel E). In the contralateral side no silver grains were detected over the neurons (Panel F). (Panels A, B, D, E, F, Bar = 10/zm; Panel C, Bar = 100 gm).


At this time point, a small number of neurons in the substantia nigra are labeled. This pattern of radioactivity is similar 2 days (Fig. 3B) and 4 days (Fig. 3C) after the injection of 125I-FGF-2, but there is a continuous and gradual decrease in silver grain density over the parenchyma that is concomitant with an increase of radioactivity over blood vessels (Figs. 3B, 3C, and 3D). By the seventh day, the total amount of radioactivity is concentrated in the scar tissue and its surrounding microvasculature (Fig. 3D).

Not all radioactivity remains directly at the site of injection. For example, 4 h after the intrastriatal injection of 125I-FGF-2, there is a dense accumulation of radioactivity over ependymal cells ipsilateral to the injected striatum (Fig. 4A). At this time, lower levels of radioactivity are detected in the ependymal cells lining the lateral ventricle on the contralateral to the side of injection, suggesting that it translocates from the parenchyma. These clusters of silver grains are still present 7 days after injection, although in lower amounts (not shown). This suggests that the FGF-2 concentrated in these structures is quite stable. Analy- sis of caudal sections reveals that all of the ependymal ceils lining the third ventricle and the central canal are also radiolabeled (not shown). Remarkably, little radioactivity is detected in the parenchyma adjacent to the ependymal layers of the ventricles, despite of the fact that there is sometimes dense labeling in the parenchyma surrounding the striatal injection site. At 7 days after the intrastriatal injection, most of the radioactivity is now associated with vessels proximal to the injection site and the corpus callosum (Figs. 4C and 4D). These findings are in stark contrast to those observed 4 h after intraventricular injection, when there is very little penetration of 125I-FGF-2 into the parenchyma adjacent to the injected ventricle. Even 4 days after this intraventricular injection, there is little intracellular clustering over ependymal cells and the silver grains localize to the apical and basal sides of ependymal ceils (Fig. 4B).

Because of the reports describing the anterograde transport of FGF-2 in various models [5,11,23,24], we also looked for radiolabeled FGF-2 at specific target cells. While the bulk of radioactivity (> 95%) is distributed as described above, some grains are found on target neurons of the substantia nigra compacta, ipsilateral to the site of injection. This select population of neurons contain small amounts of labeled FGF-2 when examined 4 h after intrastriatal injection (Fig. 4E). The quantities, though significant and reproducible, are exceedingly small compared to the distribution in other parts of the brain. In the substantia nigra, contralateral of the site of injection, the levels of autoradiographic signal were similar to the ones found in control studies (Fig. 4F).

4. Discussion

Although numerous reports have described neuro- protective effects of FGF-2 after CNS injury [3,29], our experience has been somewhat equivocal. It was for this reason that we attempted to document the fate of FGF-2 after its injection into the CNS. Several inter- esting findings are apparent from the studies that we report here. First, exogenous ~25I-FGF-2 clearly diffuses within the area of parenchymal injection but there is little diffusion into the parenchyma following an intraventricular injection. Of the total material injected, little is retrogradely or anterogradely transported from the striatum, at least at the time points and injection volumes used in this study. After a striatal injection of FGF-2, most of the radiolabel remains at the injection site, cell-associated and bound to the extracellular matrix where it is remarkably stable and presumed not to be biologically available. The striatum was selected as the site of intraparenchymal injection for its location, size and axonal projections with other brain areas. This area allowed us to analyze diffusion, binding and internalization by neuronal and non-neuronal ceils as well as the axonal transport of FGF-2. Preliminary studies indicate that the patterns of diffusion and internalization after infusion of this growth factor in other regions of the brain are similar to the ones described for the striatum (not shown). In contrast, 24 hr after nerve growth factor injection into the striatum, no radioactivity is detected in the area of injection.

After a parenchymal injection, FGF-2 accumulates very quickly (within 4 h) in the ependymal cell layer lining the ventricle. Silver grain accumulation remains dense after 4 and 7 days when SDS-PAGE analysis and autoradiography suggest that there is a slow and gradual metabolism of intact labeled FGF-2. These metabolic fragments appear identical to those gener- ated by cultured ceils after the internalization of FGF-2 [50]. This infers the presence of high affinity receptors for FGF-2.

The results also clearly suggest that a significant proportion of the ~25I-FGF-2 is internalized and metabolized by non-neuronal ceils, because a considerable number of silver grains appear over ependymal cells lining the lateral ventricle, in the corpus callosum, glial cells and over blood vessels. This finding is in agree- ment with studies that show the presence of im- munoreactive FGF-2 [10] and FGFR1 mRNA [51] in ependymal cells.

The fate of FGF-2 after injection into the CNS is in stark contrast to its fate following injection into peripheral tissues. In this latter instance, FGF-2 initially binds very tightly to the extracellular matrix, but later can be detected within mesenchymal cells and over


blood vessels at the site of injection [22]. In such non-neural tissues, FGF-2 bound to heparan sulfate in extracellular matrix (ECM) serves as a reservoir that is stable for weeks and months. In contrast, the exogenous administration of FGF-2 into the parenchyma of the brain results in its metabolism and association with blood vessels. Yet even in the brain, the injected FGF-2 is relatively long-lived and an intact FGF-2 is detected 4 days after injection. Here it is presumed to have limited bioavailability and it is sequestered in matrix and microvascular basement membranes. Its slow and gradual metabolism supports this hypothesis. This ob- servation could have a profound impact on the design and development of FGF-2-related drug delivery sys- tems in the CNS. It may also account for the variability that we have observed in establishing the neuroprotec- tive effects of FGF-2 in vivo.

Several authors have shown that FGF-2 is transported anterogradely and retrogradely in vivo [5,11,23, 24]. In the studies described here, we were more inter- ested in the fate of exogenously administered FGF-2 in surrounding tissues than in its axonal transport per se. While we did observe that 4 h after injection into the striatum, some neurons in the substantia nigra compacta appear to concentrate the radiolabeled FGF-2, a clear majority of the signal diffuses within the area of injection and along the fiber tracts of the corpus callosum. A few days after the injection, most of this radiolabeled FGF-2 is associated with non neuronal cells, especially ependymal cells lining the ventricles and central canal. This finding is in stark contrast to recent reports that exogenously administered FGF binds only to neuronal populations that express func- tional receptors t2,L3'31. While these differences may be traced to the methods used for the infusion of radiolabeled FGF-2, such as the sites of injection, dose and volume, they do agree with the known biology, bio- chemistry and biophysical characteristics of this heparin-binding growth factor.

These studies demonstrate that while the infused FGF-2 is internalized and metabolized by non-neuronal and neuronal cells, a significant proportion of the growth factor is sequestered in the extracellular matrix what is presumed to be heparan sulfate proteoglycans. This could be of particular importance for the delivery of FGFs to the brain and may explain the variable response that we have observed to FGF. Yet recent studies from Ray et al. [38] and Takayama et al. [44] indicate that the use of engineered fibroblasts produc- ing FGF-2 could represent a successful approach for FGF-2 delivery. In this paradigm, diffusion and sequestration no longer limit biological availability. Further- more their studies show that when transfected cells produce a cell-associated form of FGF-2, the best t ropic / t rophic effects are observed. These studies cor- roborate our present results and indicate that the in-

teraction of FGF-2 with heparan sulfate proteoglycans on the cell surface and matrix may serve to increase the local concentration and stability of FGF-2 in situ.

Because the exogenous FGF appears to be sequestered after its injection into the CNS, the findings described here have obvious important implications for its use as a t r o p h i c factor. N G F is retrogradely transported to cholinergic cell bodies in the medial septal nucleus [1,25,41], is not sequestered and can thus be introduced by injection or infusion at the site of axonal transection. On one hand, the increased production of matrix (i.e. scar formation) that occurs in glial response to t rauma may be a homeostatic mechanism that serves to concentrate FGF-2 at the site of injury. In this case, the administration of exogenous FGF-2 could lead to its enhanced accumulation at the site of injury and thus increased therapeutic action. On the other hand, the overproduction of matrix could serve as a sink to sequester FGF-2, limit its bioavailability and thus decrease its therapeutic action. I,n the design of appropri- ate vehicles to deliver FGF-2 [44] to the injured and diseased CNS, systematic pharmacokinetic studies will need to take into account the endogenous mechanisms that regulate its biological activity, clearance and bioavailability. To this end, the results presented here strongly suggest that a locally stable, undiffusable but biologically available formulations of FGF-2 delivery will have to be devised. Conferring the long lived characteristics of FGF-2 to other non-heparin-binding factors could also serve to enhance their in vivo action.

Acknowledgments

The authors wish to thank Annemarie P. Putze for excellent secretarial assistance in the preparat ion of this manuscript. These studies were supported by N I H DK18811, NS28121, AG06088, The Hollfelder Founda- tion, and The American Parkinson's Disease Associa- tion.

References

Ill Anderson, K.J., Dam, D., Lee, S. and Cotman, C.W., Basic fibroblast growth factor prevents death of lesioned cholinergic neurons in vivo, Nature, 332 (1988) 360-361.

[2] Bahr, M., Vanselow, J. and Thanos, S., Ability of adult rat ganglion cells to regrow axons in vitro can be influenced by fibroblast growth factor and gangliosides, Neurosci. Lett., 96 (1989) 197-201.

[3] Baird, A., Fibroblast growth factors: activities and significance of non-neurotrophin neurotrophic growth factors, Current Opin- ion in Neurobiology, 4 (1994) 78-86.

[4] Baird, A. and Bohlen, P., Fibroblast growth factors. In M.B. Sporn and A.B. Roberts (Eds.), Peptide Growth Factors, Springer-Verlag, New York, 1990, pp. 369-417.


[5] Baird, A., Morm~de, P., Ying, S.Y., Wehrenberg, W.B., Ueno, N., Ling, N. and Guillemin, R., A non-mitogenic pituitary function of fibroblast growth factor: regulation of tyrotropin and prolactin secretion, Proc. Natl. Acad. Sci. USA, 82 (1985) 5545- 5549.

[6] Baird, A., Schubert, D., Ling, N. and Guillemin, R., Receptor and heparin-binding domains of basic fibroblast growth factor, Proc. Natl. Acad. Sci. USA, 85 (1988) 2324-2328.

[7] Barotte, C., Eclancher, F., Ebel, A., Labourdette, G., Sensen- brenner, M. and Will, B., Effects of basic fibroblast growth factor (bFGF) on choline acetyltransferase activity and astroglial reaction in adult rats after partial fimbria transection, Neurosci. Lett., 101 (1989) 197-202.

[8] Barr, P.J., Cousens, L.S., Lee-Ng, C.T., Medina-Selby, A., Masiarz, F.R., Hallewell, R.A., Chamberlain, S., Bradley, J., Lee, D., Steimer, K.S., Poulter, L., Burlingame, A.L., Esch, F. and Baird, A., Expression and processing of biologically active fibroblast growth factors in the yeast Saccharomyces cerevisiae, J. Biol. Chem., 263 (1988) 16471-16478.

[9] Bikfalvi, A., Dupuy, E., Inyang, A.L., Fayein, N., Leseche, G., Courtois, Y. and Tobelem, G., Binding, internalization, and degradation of basic fibroblast growth factor in human microvascular endothelial cells, Exp. Cell Res., 181 (1989) 75-84.

[10] Cuevas, P., Gimenez-Gallego, G., Martinez-Murillo, R. and Carceller, F., Immunohistochemical localization of basic fibroblast growth factor in ependymal cells of the rat lateral and third ventricles, Acta Anat., 141 (1991)307-310.

[11] Emoto, N., Gonzalez, A.M., Walicke, P.A., Wada, E., Simmons, D.M., Shimasaki, S. and Baird, A., Identification of specifc loci of basic fibroblast growth factor synthesis in the rat brain, Growth Factors, 2 (1989) 21-29.

[12] Ferguson I.A. and Johnson, E.M.J., Fibroblast growth factor receptor-bearing neurons in the CNS: Identification by receptor-mediated retrograde transport, J. Comp. Neurol., 313 (1991) 693-706.

[13] Ferguson, I.A., Schweitzer, J.B. and Johnson, E.M., Basic fibroblast growth factor: receptor-mediated internalization, metabolism, and anterograde transport in retinal ganglion cells, J. Neurosci., 10 (1990) 2176-2189.

[14] Ferrara, N., Ousley, F. and Gospodarowicz, F., Bovine brain astrocytes express basic fibroblast growth factor, a neurotropic and angiogenic mitogen, Brain Res., 462 (1988) 223-232.

[15] Ferrari, G., Minozzi, M.-C., Toffano, G., Leon, A. and Skaper, S.D., Basic fibroblast growth factor promotes the survival and development of mesencephalic neurons in culture, Dev. Biol., 133 (1989) 140-147.

[16] Finklestein, S.P., Apostolides, P.J., Caday, C.G., Prosser, J., Philips, M.F. and Klagsbrun, M., Increased basic fibroblast growth factor (bFGF) immunoreactivity at the site of focal brain wounds, Brain Res., 460 (1988) 253-259.

[17] Florkiewicz, R.Z. and Sommer, A., Human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non-AUG codons, Proc. Natl. Acad. Sci. USA, 86 (1989) 3978-3981.

[18] Folkman, J., Klagsbrun, M., Sasse, J., Wadzinski, M.G., Ingber, D. and Vlodavsky, I., A heparin-binding angiogenic protein - basic fibroblast growth factor - is stored within basement mem- brane, Am. J. Pathol., 130 (1988) 393-400.

[19] Frautschy, S.A., Gonzalez, A.M., Martinez Murillo, R., Car- celler, F., Cuevas, P. and Baird, A., Expression of basic fibroblast growth factor and its receptor in the rat subfornical organ, Neuroendocrinology, 54 (1991) 62-67.

[20] Giulian, D., Chen, J., Ingeman, J.E., George, J.K. and Noponen, M., The role of mononuclear phagocytes in wound healing after traumatic injury to adult mammalian brain, J. Neurosci., 9 (1989) 4416-4429.

[21] Gonzalez, A.-M, Buscaglia, M., Ong, M. and Baird, A., Distri-

bution of basic fibroblast growth factor in the 18-day rat fetus: localization in the basement membranes of diverse tissues, J. Cell. Biol., 110 (1990) 753-765.

[22] Gonzalez, A.-M., Buscaglia, M.L., Fuller, J., Dahl, R., Carman, L.S. and Baird, A., Local fate and distribution of locally infused basic FGF: the example of the rat brain and the Xenopus tail mesenchyme. In A. Baird and M. Klagsbrun (Eds.), Fibroblast Growth Factors, Annals of the New York Academy of Sciences, 1991, pp. 416-419.

[23] G6mez-Pinilla, F., Lee, J.W.-K. and Cotman, C.W., Basic FGF in adult rat brain: cellular distribution and response to enthori- nal lesion and fimbria-fornix transection, J. Neurosci., 12 (1992) 345-355.

[24] Johnson, D.E., Lee, P.E. and Williams, L.T., Diverse forms of a receptor for acidic and basic fibroblast growth factors, Mol. Cell. Biol., 10 (1990) 4728-4736.

[25] Johnson, E.M, Taniuchi, M., Clark, H.B., Springer, J.E., Koh, S., Tayrien, M.W. and Loy, R., Demonstration of the retrograde transport of nerve growth factor receptor in the peripheral and central nervous system, J. Neurosci., 7 (1987) 923-929.

[26] Kardami, E. and Fandrich, R.R., Basic fibroblast growth factor in atria and ventricles of the vertebrate heart, J. Cell Biol., 109 (1989) 1865-1875.

[27] Lipton, S.A., Wagner, J.A., Madison, R.D. and D'Amore, P.A., Acidic fibroblast growth factor enhances regeneration of pro- cesses by postnatal retinal ganglion cells in culture, Proc. NatL Acad. Sci. USA, 85 (1988) 2388-2392.

[28] Logan, A., Acidic and basic fibroblast growth factor genes show individual time-related responses following central nervous system lesion, J. EndocrinoL, 124 (1990) 47-40.

[29] Logan, A. and Berry, M., Transforming growth factor-bl and basic fibroblast growth factor in the injured CNS, Trends Pharm- col. Sci., 14 (1993) 337-343.

[30] Logan, A., Frautschy, S.A., Gonzalez, A.-M. and Baird, A., A time course for the focal elevation of synthesis of basic fibroblast growth factor and one of its high-affinity receptors (fig) following a localized cortical brain injury, J. Neurosci., 12 (1992) 3828-3837.

[31] McGeer, E.G., Singh, E.A. and McGeer, P.L., Apparent anterograde transport of basic fibroblast growth factor in the rat nigrostriatal dopamine system, Neurosci. Lett., 148 (1992) 31-33.

[32] McNeil, P.L., Muthukrishnan, L., Warder, E. and D'Amore, P.A., Growth factors are released by mechanically wounded endothelial cells, J. Cell Biol., 109 (1989) 811-822.

[33] Moscatelli, D., Metabolism of receptor-bound and matrix-bound basic fibroblast growth factor by bovine capillary endothelial cells, J. Cell Biol., 107 (1988) 753-759-753-750.

[34] Nieto-Sampedro, M., Lim, R., Hicklin, D.J. and Cotman, C.W., Early release of glia maturation factor and acidic fibroblast growth factor after rat brain injury, Neurosci. Left., 86 (1988) 361-365.

[35] Otto, D., Frotscher, M. and Unsicker, K., Basic fibroblast growth factor and nerve growth factor administered in gel foam rescue medial septal neurons after fimbria fornix transection, J. Neu- rosci. Res., 22 (1989) 83-91.

[36] Otto, D. and Unsicker, K., Basic FGF reverses chemical and morphological deficits in the nigrostriatal system of MPTP- treated mice, J. Neurosci., 10 (1990) 1912-1921.

[37] Otto, D., Unsicker, K. and Grothe, C., Pharmacological effects of nerve growth factor and fibroblast growth factor applied to the transectioned sciatic nerve on neuron death in adult rat dorsal root ganglia, Neurosci. Lett., 83 (1987) 156-160.

[38] Ray, J., Hogg, J., Beutler, A.S., Takayama, H., Baird, A. and Gage, F.H., Expression of biologically active basic fibroblast growth factor by genetically modified rat primary skin fibroblasts, J. Neurochem., (1994) in press.

[39] Rogelj, S., Klagsbrun, M., Atzmon, R., Kurokawa, M.,


Haimovitz, A., Fuks, Z. and Vlodavsky, I., Basic fibroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiation of PCI2 cells, J. Cell Biol., 109 (1989) 823-831.

[40] Rogister, B., Leprince, P., Pettmann, B., Labourdette, G., Sensenbrenner, M. and Moonen, G., Brain basic fibroblast growth factor stimulates the release of plasminogen activators by newborn rat cultured astroglial cells, Neurosci. Lett., 91 (1988) 321-326.

[41] Shimasaki, S., Emoto, N., Koba, A., Mercado, M., Shibata, F., Cooksey, K., Baird, A. and Ling, N., Complementary DNA cloning and sequencing of rat ovarian basic fibroblast growth factor and tissue distribution study of its mRNA, Biochem. Biophys. Res. Comrnun., 157 (1988)256-263.

[42] Shults, C.W., Yajima, H., Gullner, H.G., Chase, T.N. and O'Donohue, T.L., Demonstration and distribution of Kassinin- like material (Substance K) in the rat central nervous system, J. Neurochem., 45 (1994) 445-558.

[43] Sievers, J., Havsmann, B., Unsicker, K. and Berry, M., Fibrob- last growth factors promote the survival of adult retinal ganglion cells after transections of the optic nerve, Neurosci. Lett., 76 (1987) 157-162.

[44] Takayama, H., Ray, J., Baird, A., Beutler, A.S. and Gage, F.H., Intracerebral grafting of cells genetically modified to express basic fibroblast growth factor, Soc. Neurosci. Abstr., 18 (1992).

[45] Thompson, J.A., Anderson, K.D., DiPietro, J.M., Zwiebel, J.A., Zametta, M., Anderson, W.F. and Maciag, T., Site-directed neovessel formation in vivo, Science, 241 (1988) 1349-1352.

[46] Unsicker, K., Reichert-Preibsch, H., Schmidt, R., Pettmann, B., Labourdette, G. and Sensenbrenner, M., Astroglial and fibroblast growth factors have neurotrophic functions for cultured peripheral and central nervous system neurons, Proc. Natl. Acad. Sci. USA, 84 (1987) 5459-5463.

[47] Vlodavsky, 1., Folkman, J., Sullivan, R., Fridman, R., Ishai- Michaeli, R., Sasse, J. and Klagsbrun, M., Endothelial cell-de- rived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix, Proc. Natl. Acad. Sci. USA, 84 (1987) 2292-2296.

[48] Walicke, P., Cowan, M.W., Ueno, N., Baird, A. and Guillemin, R., Fibroblast growth factor promotes survival of dissociated hippocampal neruons and enhances neurite extension, Proc. Natl. Acad. Sci. USA, 83 (1986) 3012-3016.

[49] Walicke, P.A. and Baird, A., Neurotrophic effects of basic and acidic fibroblast growth factors are not mediated through glial cells, Deu. Brain Res., 40 (1988) 71-79.

[50] Walicke, P.A. and Baird, A., Internalization and processing of basic fibroblast growth factor by neurons and astrocytes, J. Neurosci., 11 (1991) 2249-2258.

[51] Wanaka, A., Johnson, E.M.J. and Milbrandt, J., Localization of FGF receptor mRNA in the adult rat central nervous system by in situ hybridization, Neuron, 5 (1990) 267-281.

[52] Weiner, H.L. and Swain, J.L., Acidic fibroblast growth factor mRNA is expressed by cardiac myocytes in culture and the protein is localized to the extracellular matrix, Proc. Natl. Acad. Sci. USA, 86 (1989) 2683-2687.

Documents

Storage, metabolism, and processing of 125I-fibroblast growth factor-2 after intracerebral injection