Increased basic fibroblast growth factor mRNA following contusive spinal cord injury

Molecular Brain Research, 22 (1994) 1-8

1

© 1994 Elsevier Science B.V. All rights reserved 0169-328X/94/$07.00

BRESM 70701

Research Reports

Increased basic fibroblast growth factor mRNA following contusive spinal cord injury Paolo Follesa, Jean R. W r a t h a l l , I t a l o M o c c h e t t i * Department of Anatomy and Cell Biology, Georgetown University, Medical School, 3900 Reservoir Rd, N.W., Washington, DC 20007, USA (Accepted 27 July 1993)

Key words: Basic fibroblast growth factor mRNA; Acidic fibroblast growth factor; Nerve growth factor; trkA; Rat; RNase protection assay

Neurotrophic factors appear to be crucial for the survival and potential regeneration of injured neurons. Injury of the peripheral nervous system results in the induction of a number of neurotrophic molecules. Less is known about the response of central nervous tissue to injury. We have examined changes in levels of mRNA for three trophic factors, basic and acidic fibroblast growth factor (bFGF, aFGF), and nerve growth factor (NGF), after a standardized incomplete thoracic contusive spinal cord injury (SCI). RNase protection assays showed a rapid increase (3-fold) in the content of bFGF mRNA by 6 hours after SCI in tissue that included the injury site. No effect of injury was seen in segments of cervical or lumbar cord. bFGF mRNA at the injury site remained significantly increased at 1 and 7 days after SCI. Further, at 7 days, the increase was anatomically restricted to the rostral portion of the injury site suggesting the involvement of specific pathways in the maintenance of high levels of bFGF mRNA. No change in the levels of aFGF mRNA was seen after SCI. Similarly, no difference in the expression of the mRNA for NGF or its high affinity receptor (trkA), were observed at 6 h, 1 or 7 days following SCI. Our observation of a specific effect of SCI on bFGF mRNA expression supports a speculative hypothesis that bFGF may play a role in the partial recovery of function seen following incomplete contusive spinal cord injury.

INTRODUCTION

The adult mammalian central nervous system (CNS) fails to regenerate nerve fibers after injury. However, after an incomplete spinal cord injury (SCI), some recovery typically occurs as demonstrated by the functional improvement over time shown both in experimentally injured animals 2°'26'34'56 and in patients after SCI 3. Little is known about the basis of such partial recovery after SCI. However, it seems obvious that an understanding of the basis for even a limited amount of natural recovery could lead to opportunities to therapeutically enhance such processes. In the peripheral nervous system (PNS), recovery from injury to peripheral nerves occurs via actual regeneration of injured axons. This, in turn, is supported by the induction of trophic factors, such as nerve growth factor (NGF), and its receptor in Schwann cells of the axotomized distal nerve stump 24. Injury to the CNS can also lead to increases in factors with neurotrophic properties. Among these is basic fibroblast

* Corresponding author. Fax: (1) (202) 687-1823.

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growth factor (bFGF), a protein that is normally synthesized in brain and spinal c o r d 6'13A5,42. bFGF has been shown to have a potent effect on the survival and proliferation of CNS glia and endothelial cells, as well as on the survival and outgrowth of CNS neurons in v i t r o 16'28'33'45'51'52 and in v i v o 1'36'37. Recent studies have shown that bFGF immunoreactivity is markedly increased in the area surrounding a focal cortical brain wound at one week after injury 12't8. The bFGF is thought to be important for the proliferation of capillary endothelial cells 5,23 including that occurring during the first two weeks after injury that results in neovascularization of the lesioned area 45. However, bFGF may have additional functions. In the adult CNS, the application of exogenous bFGF has been shown to rescue specific populations of axotomized cholinergic neurons and to promote their fiber regrowth 1,36. Thus, increases in endogenous bFGF could be important for the survival and of injured spinal cord neurons and the limited recovery that is seen. Because of its properties, bFGF may therefore play an important role in the cascade of cellular changes following mammalian spinal cord injury. In this report, we present the first evidence that SCI rapidly induces bFGF expression and that this

expression remains significantly enhanced for at least 1 week after injury. MATERIALS AND METHODS

Spinal cord injury Female Sprague-Dawley rats (200-220 g) were anesthetized with chloral hydrate, a laminectomy performed at the T s vertebral level and a 10 g weight was dropped 2.5 cm onto exposed dura, as previously described54. The biomechanics of this injury model 3s as well the behavioral, neurophysiological and histopathological consequences of injury have been described2°'26"34'35'39. Controls received a laminectomy with the injury device lowered onto the dura but no weight was dropped. Rats were sacrificed by decapitation at the specified times after injury. Spinal cords were quickly removed and dissected into cervical, thoracic and lumbar sections. The thoracic section was divided at the injury site into two 15 mm segments, rostral and caudal to the contusion site, and tissue from the cervical (20 mm) and lumbar (15 mm) enlargements also removed. Tissues were immediately frozen on dry ice and kept at - 70°C until analysis.

Probe preparation The plasmid RObFGF103 (a gift from Dr. A. Baird, The Whittier Institute, La Jolla, CA) is a derivative of a bluescript plasmid containing a 1016 base portion of the rat bFGF cDNA47. NeoI linearized plasmid was used as a template for the in vitro transcription assay with T7 RNA polymerase42. This procedure generated a 32p-labeled 524 base probe which includes the 477 bases of bFGF cRNA and 47 bases of the bluescript polylinker region. HBGF-1 plasmid (a gift from Dr. Goodrich, Alton Jones Cell Science Center, Lake Placid, NY) containing the cDNA encoding for rat acidic fibroblast growth factor (aFGF) 22, was linearized with NcoI and used as a template with the T3 RNA polymerase to generate a 662 base of aFGF 32p-labeled cRNA comprising 44 bases of the bluescript polylinker region. 32P-Labeled NGF RNA probe was generated from plasmid BSrNGF 53 which contains a 771 base portion of the rat NGF cDNA (a gift from Dr. Whittemore, University of Miami, Miami, FL). This plasmid was used after linearization with EcoRI as a template for the in vitro transcription assay with T3 RNA polymerase. This procedure generated a 32p-labeled 830 base probe which includes the 771 bases of NGF cRNA and 59 bases of the bluescript polylinker region. The 760 base BamHI-EcoRI fragment containing the 5' end eDNA encoding rat trkA was isolated from pDMll53° and subcloned into a pGEM-7Z vector (Promega) to generate plasmid pCAlt5 l°. EcoRl linearized pCAll5 was used as a template for the in vitro transcription assay with SP6 RNA polymerase. This procedure generated a 32p-labeled 805 base probe which include the 760 bases of trkA cRNA and 45 bases of the pGEM-7Z polylinker region. Plasmid pGI1542, a derivative of plasmid plB15, contains a partial sequence of the rat cyclophilin cDNA11 encoding for a constitutive protein. The in vitro transcription with SP6 polymerase of the EcoRI linearized pGI15 generated a cRNA composed by 294 bases complementary to cyclophilin RNA and 7 bases of the plasmid polylinker region. Cyclophilin cRNA was used as standard control to monitor for artifacts due to extraction of RNA from tissue sample or differences in loading in each experiment.

RNase protection assay RNase protection assay was carried out as previously described42. Briefly, total RNA was extracted from brain tissues as described elsewhere7. RNA (20-25/zg) was dissolved in 20/zl of hybridization solution and containing 150,000 cpm of a 32P-labeled neurotrophic factor cRNA probe (spec. act. > 6 × 108 cpm/p,g of RNA each). To balance the relative high abundance of cyclophilin RNA, piG15 was labeled to a lower specific activity ( ~ 1 × 106 cpm/p,g of RNA). Hybridization was carried out at 50°C overnight. RNA was digested

with RNase A (1 U/ml) and T1 (200 U/ml) for 30 minutes at 35°C. The reaction was stopped by extracting the sample with a solution (1 : 1 v/v) containing 4 M guanidine isothiocyanate, 25 mM sodium citrate, 0.5 M sarkosyl, and 0.1 M 2-mercaptoethanol and samples were ethanol precipitated. The pellet containing the RNA:RNA hybrid was dissolved in loading buffer (80% formamide, 0.1% xylene cyanol, 0.1% Bromophenol blue, 2 mM EDTA), boiled at 95°C and separated on a 5% polyacrylamide/urea sequencing gel. 32p-Endlabeled (T4 polynucleotide kinase) Mspl digested pBR322 fragments were used as a molecular marker. The gel was dried and the bFGF, aFGF and NGF mRNA protected fragments were visualized by autoradiography on X-ray film using Chronex Quanta III intensifying screen.

RNA calculation Total RNA was quantified by absorption at 260 nm. Neurotrophic factor mRNA content was calculated by measuring the peak densitometric area of the autoradiograph analyzed with a laser densitometer (Hoefer GS 300 scanning densitometer) normalized by the peak densitometric area of the cyclophilin autoradiograph band. The exposure time and amount of RNA needed for the quantification were predetermined previously by measuring bFGF, and aFGF autoradiographs plotted vs a standard curve obtained by loading on the gel increasing amount of RNA to assure that the autoradiograph bands were in the linear range of intensity. The values are expressed as% of control (laminectomized) animals and are the mean + S.E.M.

Statistical analysis Normality test was used to verify the homogeneity of the values. Differences among means were evaluated by ANOVA. When treatment elicited significant changes, significance was determined by Dunnett's test (for comparing treatment groups with control group) and Sheffe's test (for multiple comparisons).

RESULTS

Expression o f neurotrophic factor m R N A s in adult spinal cord

The RNA protection assays allowed direct comparison of the mRNA levels for bFGF, aFGF, NGF and the trkA (NGF high-affinity) receptor in the same tissue samples. Consistent high levels of expression of bFGF mRNA were seen in spinal cords of laminectomy control rats (Fig. 1) as expected in adult spinal cord tissue 42. Levels of aFGF were even higher than those of bFGF (% values for aFGF mRNA 245 + 49, mean + SEM vs bFGF mRNA), confirming the observation that aFGF is highly expressed in adult spinal cord 5°. In contrast, very low levels of mRNA for NGF and trkA were seen, necessitating a much longer exposure of the autoradiographs to estimate their mRNA levels (Fig. 2).

bFGF mRNA induction in spinal cord injury In control rats, 7 days after a laminectomy at the T 8 vertebral level, the content of bFGF mRNA was similar in different segment of the spinal cord, including the cervical and lumbar enlargements and both the rostral and caudal samples of the thoracic cord that had been divided at the laminectomy site (data not shown). In contrast, in tissue 7 days after SCI, we

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P

aFGF bFGF

250 "~ 200 to

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bFGF

150 100

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Fig. 1. Detection of aFGF and bFGF mRNA levels in control and injured spinal cord with the RNase protection assay. A mild contusive injury at T 8 was produced by a weight drop technique in female rats. Rat were killed by decapitation 7 days after the lesion and RNA was extracted from the thoracic segment rostral to the injury site as described in Materials and Methods. 25/~g of RNA were analyzed by RNase protection assay as described in Materials and Methods. The bands of bFGF or aFGF mRNA protected fragments (477 and 618 bases, respectively) are shown. Arrows indicate the bFGF and aFGF mRNA unprotected fragments (524 and 662 bases, respectively). L, laminectomized; I, injured; T, tRNA; P, digested probe (aliquot of the hybridization solution containing the cRNA probe for bFGF and aFGF ~ 3,000 cpm). The autoradiographic film was exposed for 24 h at - 70°C with an intensifying screen.

consistently found a significant increase ( ~ 2-fold) in bFGF mRNA content in the rostral but not in the caudal thoracic segments that included the injury site (Figs. 1 and 3). No changes in bFGF mRNA levels were detected in the cervical or lumbar spinal segments. In order to compare the changes in the content

PT

D

L

I

L

I

trkA

aFGF

bFGF

Fig. 2. Detection of trkA mRNA in control and injured spinal cord. Tissue samples were prepared as indicated in the legend of Fig. 1. RNA was extracted from the thoracic segment rostral to the injury site. 35 /zg of total RNA were used. trkA (730 bases), aFGF and bFGF RNA-protected fragments are indicated. P, aliquot of the hybridization solution containing the cRNA probe for trkA (810 unprotected bases), bFGF and aFGF ~ 3,000 cpm (arrows indicate the unprotected fragments); T, tRNA; D, digested probe; L, laminectomy; I, injury. The autoradiographic film was exposed for 6 days at -70"C with an intensifying screen. In addition, to demonstrate the trkA bands in the figure, that portion of the negative was photographically exposed longer.

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Fig. 3. bFGF mRNA increases in the thoracic segments following SCI. bFGF and aFGF mRNA levels were determined in thoracic, lumbar and cervical segments of spinal cord injured at thoracic levels, 7 days after injury. The amount of bFGF and aFGF mRNA was calculated by dividing the peak densitometric area of the bFGF or aFGF mRNA autoradiographic band by the amount of cyclophilin mRNA. Data are the mean + S.E.M. of three separate experiments, with at least 3 rats per group each experiment (n = 9). * * P < 0.01.

of bFGF mRNA with other trophic factors known to be induced following brain injury, we simultaneously assayed mRNA encoding for aFGF in the identical tissue extracts. Although aFGF mRNA is highly expressed in adult spinal cord, SCI failed to affect aFGF mRNA levels in either the rostral or caudal segments containing the injury site or the cervical or lumbar enlargements (Figs. 1 and 3). Moreover, in confirmation of our previous report 4, the content of cyclophilin mRNA did not change following SCI.

Temporal changes in bFGF mRNA following SCI It has been established that in human spinal cord trauma a rapid pharmacological intervention (within 8 h) is necessary for enhancing recovery of function 3, suggesting the importance of events occurring shortly after SCI. To determine whether injury rapidly produces changes in bFGF mRNA levels, we examine tissue extracts at 6 and 24 h after SCI. A significant increase in bFGF mRNA was observed at 6 h after SCI in the rostral segment adjacent to the injury site (Fig. 4), with a lesser, and statistically insignificant increase in the caudal thoracic segment. At 1 day, bFGF mRNA was significantly elevated in both the rostral and caudal thoracic segments, but continued to show the largest increase in the former. In the cervical and lumbar segments bFGF mRNA failed to change at any time (data not shown). Therefore, the enhanced expression of bFGF mRNA appears to be limited to the lesioned region and/or adjacent tissue rather than representing

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350

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300 2so 20O

150 100 6 hr

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Fig. 4. Temporal changes in b F G F m R N A following spinal cord injury in the thoracic segment. Rat were subjected to a mild contusive injury and killed at the indicated time. b F G F m R N A was measured in the thoracic segment. The a m o u n t of b F G F m R N A detected was calculated by dividing the peak densitometric area of the b F G F m R N A autoradiographic band by the a m o u n t of cyclophilin m R N A . The results, expressed as percent of control animals, represent the m e a n + S . E . M , of 3 independent experiments, each with 3 replicate tissue samples (n = 9). * P < 0.05, * * p < 0.0l.

a diffuse response throughout the spinal cord. In contrast to the effect of SCI on bFGF, the levels of a F G F m R N A were maintained at control levels at 6 and 24 h after SCI in all segments (data not shown).

NGF and trkA mRNA failed to change in injured spinal cord We have previously showed that the low affinity N G F receptor m R N A (p75 N~FR) increases in the site of the lesion and in the surrounding tissue 4'4°. It was therefore of interest to determine whether N G F and its high-affinity receptor, trkA, m R N A can be found in [ ] NGF I I trkA 125

"6 100 c o o

75 50

25

0 6 hr

1 day 7 days

Fig. 5. trkA and NGF mRNA levels fail to change following SCI. Spinal cord was injured as described, and total RNA was extracted from the thoracic segments rostral or caudal to the lesion site. Rats were sacrificed at the indicated time. Data of 6 h and 1 day are the mean 5: S.E.M. of three separate experiments (n = 9); data of 7 days are the mean + S.E.M. of 6 different tissue samples.

spinal cord and, if so, whether their expression is altered in response to the injury. N G F m R N A was barely detectable in control rats confirming the hypothesis that adult spinal cord does not synthesize large amounts of NGF. Moreover, neither laminectomy nor injury induced N G F m R N A expression to higher levels under our experimental condition (Fig. 5). Similarly, only very low levels of trkA m R N A could be detected in the different segments of the spinal cord and the levels remained similar at 6 h, l and 7 days following SCI (Figs. 2 and 5) in contrast to p75 N6FR m R N A which has been shown to increase several fold at the injury site within 4 days after SCI 4. These data suggest that both the synthesis and biological activity of N G F in the adult spinal cord injury is limited in both the normal and injured spinal cord. DISCUSSION Our results demonstrate a rapid increase in the levels of m R N A for b F G F in the spinal cord by 6 h after a traumatic injury that persists for at least 1 week after SCI. Further, the effect on b F G F m R N A is specific, with no significant increase in a F G F or N G F m R N A . There are several ways in which such a up-regulation of b F G F m R N A could be involved in the partial recovery of function that is seen in this model of SC120,35.54. One mechanism through which b F G F could support recovery is by stimulation of neovascularization. It has been suggested that brain injury, such as occurs after stroke or head trauma, increases availability of b F G F and thereby activates a cascade of events that culminate in the production of glial scars and neovascularization 18. Hence, by analogy to peripheral tissue where b F G F plays a role in wound healing 32'41, b F G F could likewise participate in the repair of CNS tissue. Traumatic spinal cord injury results in vascular injury, hemo r r h a g e , r e d u c e d b l o o d flow and c o n s e q u e n t ischemia 49. For continued survival as well as recovered function, tissue that has been spared by the initial trauma must be revascularized. Re-establishment of blood supply requires proliferation of vascular endothelial cells, a process that is stimulated by bFGF. We have previously demonstrated a network of blood vessels traversing the injury site by 1 week in this injury model 4°. The increase in b F G F m R N A at the injury site at 6 h and 1 day after injury, assuming it is reflected in increased b F G F protein released during the tissue degeneration that occurs after SCI, would support the observed neovascularization. Thus, our data, showing that increased b F G F m R N A is an early event following SCI, supports the current view that

availability of bFGF is important for tissue repair and might be involved in the neovascularization process. A second mechanism could be through bFGF acting as a neurotrophic factor, bFGF is a potent neurotrophic factor in vitro, supporting neuronal survival, neurotransmitter synthesis and neurite outgrowth in a variety of neurons 28'33'51'52. The broad range of neurons whose survival is supported by bFGF suggests that it could be a general neuron survival-promoting factor. In vivo, infusion of exogenous bFGF has been shown to increase cholinergic neuron survival after fimbria-fornix transection TM, and decrease loss of dopaminergic neurons following MPTP infusion37. Survival of axotomized spinal cord neurons may also be enhanced by an increase in endogenous bFGF levels after SCI. bFGF synthesized in the CNS appears to be necessary for the maturation of central cholinergic and dopaminergic neurons since it stimulates choline acetyltransferase activity in septal cultures and enhances dopamine uptake in mesencephalic cultures 16'28'36. Thus, neurotransmitter synthesis in surviving neurons may be enhanced by increased levels of bFGF. Our data allow the speculation that the partial recovery of choline acetyltransferase activity, the rate-limiting enzyme in the synthesis of acetylcholine, at the injury site, that we see beginning at 2 weeks post-injury 2 might be stimulated by increased levels of bFGF. Spinal cord trauma initiates secondary injury processes that contribute to the overall tissue damage and functional impairment, including increases in concentrations of excitatory amino acids (EAA) that have been associated with neurotoxicity9. The effect of antagonists of EAA in reducing the consequences of SCI in this 55'56 and similar models strongly suggests that excitotoxic phenomenon contribute significantly to the injury process. Thus, it is noteworthy that exogenous bFGF has also been shown to limit hippocampal neuronal death caused by glutamate, most likely by raising the threshold for glutamate neurotoxicitys. bFGF also antagonizes the outgrowth-inhibiting actions of glutamate on hippocampal neurons 31, suggesting that bFGF may have potential for preventing, or enhancing recovery from, excitotoxic damage. The increase in endogenous bFGF could provide a natural mechanism to reduce the effects of abnormal EAA release after SCI and support functional recovery through stimulation of neurotransmitter synthesis and sprouting of surviving neurons. Through one or more of these mechanisms the rapid induction of bFGF following SCI could support the partial recovery of function that subsequently occurs, provided that increased bFGF mRNA is correlated with increased levels of bFGF protein that is available

to target cells. We have not yet measured bFGF protein levels in this injury model. However, in the adult brain mechanical injury has been associated with increased bFGF and aFGF immunoreactivity at 7 days after injury at the margin of the injury site 18. Further, in another model of incomplete thoracic SCI, where vascular stasis is produced with a photochemical lesion, bFGF immunoreactivity was shown to be increased 5 and 12 days later 29. The increased bFGF immunoreactivity was seen in reactive astrocytes near the injury site and also in tissue 1 cm rostral to the injury site at the T4-5 level of the spinal cord. A similar increase in samples of tissue 1 cm caudal to the lesion was not found. Our finding of increased bFGF mRNA levels at 7 days after injury in samples that included the epicenter as well as tissue up to 1.5 cm rostral to it would be consistent with such a distribution. However, localization of bFGF protein by immunocytochemistry in our model, and correlative in situ hybridization to localize mRNA will be needed to establish the cellular basis of the induction we have observed. Our observation of a greater induction of bFGF mRNA rostral to the lesion combined with the report of enhanced bFGF immunoreactivity rostral to a spinal cord lesion 29 suggests the involvement of mechanisms in addition to the local response at the injury site. The data favor an hypothesis that molecular events linked to the neuronal wiring of specific spinal cord pathways are involved in the up-regulation of bFGF mRNA at day 7. In contrast, at day 1 there is also a significant induction in the samples containing the epicenter and tissue caudal to it. Our speculation is that there is a transient induction of bFGF mRNA at the injury site due to the mechanical injury as well as a more pronounced and long-lasting induction rostral to the injury site that is related to events specific to the tissue rostral to the injury. In our model of SCI where there is widespread gray matter pathology and probably damage to at least some axons in virtually all spinal pathways34'35, a preferential induction in the rostral tissue segments could most easily be explained in relationship to particular ascending or descending tracts. For example, Wallerian degeneration of ascending pathways might preferentially induce bFGF in the dorsal columns, as has been observed after a photochemical lesion z9. Alternatively, or in addition, abnormal release of particular neurotransmitters from specific descending tracts that are injured might stimulate bFGF mRNA rostral to the injury site. This would be in line with our observation that synaptic activity may induce bFGF mRNA aside from the mechanisms associated with mechanical injury 43. These hypotheses will be tested in future experiments.

Although significant and persistent increases in b F G F m R N A were found from 6 h through 7 days after SCI, we found no effect of injury on levels of m R N A for al~GF. The absence of increases in a F G F m R N A is consistent with the interpretation of increased a F G F immunoreactivity in photochemically lesioned spinal cord as being due to accumulation of anterogradely transported protein 29. Similarly, we found no induction of N G F m R N A after SCI. These data suggest that the increase in NGF-like immunoreactivity we previously described at 7 days after injury 2, was also due to accumulation of protein synthesized outside of the spinal cord. N G F binds with high affinity to the product of the trk proto-oncogene, a 140 kDa protein with tyrosine kinase activity. This protein, called t r k A , is now believed to be the high affinity receptor for N G F and to be necessary for its biological activity 25'27. Our data from this study indicate that SCI also fails to induce t r k A mRNA. Although responses to NGF, independent from activation of a rapid signal transduction mechanism, could participate in late events that are also important for tissue repair, our data suggest that N G F is unlikely to be therapeutic for SCI. Indeed, in the adult spinal cord N G F appears to be devoid of any significant effect in rescuing neurons from cell death 57. The absence of t r k A induction is likely the main reason for such unresponsiveness. On the other hand, we have previously shown that p75 NcvR m R N A is induced following SCI 4'40. The expression of p75 NGFR m R N A has also been shown to occur in PNS and other areas of the CNS when injured. Indeed, the low affinity N G F receptor is produced in Schwann cells in response to axotomy 24 and in the CNS following a focal injury 14'19'21. In addition to NGF, other members of the neurotrophin family, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), can bind p75 NGFR44,48. It has been suggested that availability of p75 NGvR might allow for more efficient retrograde transport of neurotrophins to the neuronal cell body where neurotrophins can modulate biosynthesis and activities of a wide variety of proteins ultimately affecting events responsible for plasticity and modulation of neuronal function. Thus, the low affinity receptor might be involved in cell-cell interaction and in events important for cell maintenance. Therefore, expression of p75 NGFR in injured cells may serve to concentrate at the cell surface other members of the neurotrophin family thereby guiding and promoting regeneration of neurotrophin-responsive neurons. This might be in line with the recent observation that B D N F and to a lesser extent NT-3, rescue spinal cord motoneurons from

axotomy-induced cell death in developing rats~7. Thus, after lesion, one might expect to see changes in B D N F levels rather than NGF, to lead to rescue of spinal motoneurons. Experiments to test this hypothesis are in progress. Injury of PNS and CNS, induces biological events that trigger varying degrees of recovery. With certain types of injury in the PNS recovery may include actual regeneration of injured axons. This in turn is supported by cellular and molecular events that involve interaction between neurons and glia, and the production of neurotrophic molecules. In the CNS, regeneration per se does not appear to occur. Nevertheless, in experimental models of CNS traumatic injury, there is often considerable recovery of function as compared to acutely profound functional deficits. Little is currently known about the molecular basis for this recovery, but it is reasonable to hypothesize that neurotrophic molecules may be involved. Our results support this hypothesis by demonstrating increased m R N A levels for a molecule, bFGF, that has neurotrophic properties as well as a broad action in wound healing. Acknowledgements. The authors wish to thank Drs. A. Baird, S.

Whittemore, S.P. Goodrich, M. Chao, D. Martin-Zanca, L. Parada, R. Milner for the gift of plasmids. Special thanks to Dr. M. Riva for its invaluable help with the first experiment. This work was supported by American Paralysis Association Grant MB1-9104-1, and by H.H.S. Grants NS 29664 and NS 28130. REFERENCES 1 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 Bakhit, C., Armanini, M., Wong, W.L.T., Bennett, G.L. and Wrathall, J.R., Increase in nerve growth factor-like immunoreactivity and decrease in choline acetyltransferase activity following contusive spinal cord injury, Brain Res., 554 (1991) 264-271. 3 Bracken, M.B., Shepard, M.J., et al., A randomized controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury, New Engl. J. Med., 322 (1990) 1405±1411. 4 Brunello, N., Reynolds, M., Wrathall, J.R. and Mocchetti I., Increased nerve growth factor receptor mRNA in contused spinal cord, Neurosci. Lett., 118 (1990) 238-240. 5 Burgess, W. and Maciag, T., The heparin-binding growth factor family of proteins, Annu. Rev. Biochem., 58 (1989) 575-590. 6 Caday, C.G., Klagsburgn, M., Fanniong, P.J., Mirzabegian, A. and Finklestein, S.P., Fibroblast growth factor levels in the developing rat brain, Dev. Brain Res.,, 52 (1990) 241-246. 7 Chomczynski, P. and Sacchi, N., Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroformextraction, Anal Biochem., 162 (1987) 156-159. 8 Cheng, B. and Mattson, M.P., NGF and bFGF protect rat hippocampal and human cortical neurons against hypoglycemicdamage by stabilizing calcium homeostasis, Neuron, 7 (1991) 10311041. 9 Choi, D.W., Glutamate neurotoxicity and diseases of the nervous system, Neuron, 1 (1988) 623-634. 10 Colangelo, A.M., Fink, D. and Moccheni, I., Induction of nerve growth factor responsiveness in C6-2B glioma cells by expression of trkA proto-oncogene,Glia, submitted.

11 Danielson, P.E., Forss-Petter, S., Brown, M.A., Calavetta, L., Douglass, J., Milner, R.J. and Sutcliffe, G.J., plBl5: a eDNA clone of the rat mRNA encoding cyclophilin, DNA, 4 (1988) 261-267. 12 Eckenstein, F.P., Shipley, G.D. and Nishi, R., Acidic and basic fibroblast growth factor in the nervous system: distribution and differential alteration of levels after injury of central versus peripheral nerve, J. Neurosci., 11 (1991) 412-419. 13 Emoto, N., Gonzales, A.-M., Walicke, P.A., Wada, E., Simmons, D.M., Shimasaki, S. and Baird, A., Basic fibroblast growth factor in the central nervous system: identification of specific loci of basic FGF expression in the rat brain, Growth Factors, 2 (1989) 21-29. 14 Ernfors, P., Henschon, A., Olson, L. and Persson, H., Expression of nerve growth factor receptor mRNA is developmental regulated and increased after axotomy in rat spinal cord motoneurons, Neuron, 2 (1989) 1605-1613. 15 Ferrara, N., Ousley, F. and Gospodarowicz, D., Bovine brain astrocytes express basic fibroblast growth factor, a neurotrophic and angiogenic mitogen, Brain Res., 462 (1988) 223-32. 16 Ferrari, G., Minozzi, M.C., Toffano, G., Leon, A. and Skaper, S.D. Basic fibroblast growth promotes the survival and development of mesencephalic neurons in culture, Dev. Biol., 133 (1989) 140-147. 17 Frisen, J., Verge, M.K., Cullheim, S., Fried, K., Middlemans, D.S., Hunter, T., H6kfelt, T. and Risling, M. Increased levels of trkB mRNA and trkB protein-like immunoreactivity in the injured rat and cat spinal cord, Proc. Natl. Acad. Sci. USA, 89 (1992) 11282-11286. 18 Finklestein, P., Apostolides, P.J., Caday, C.G., Prosser, J., Philips, M.F. and Klagsbrun, M., Increased basic fibroblast growth factor immunoreactivity at the site of focal brain wound, Brain Res., 460 (1988) 253-259. 19 Gage, F.H., Batchelor, P., Chen, K.S., Chin, D., Higgins, G.A., Koh, S., Deputy, S., Rosenberg, M.B., Fisher, W. and Bj6rklund A., NGF receptor reexpression and NGF-mediated cholinergic neuronal hypertrophy in the damaged adult neostriatum, Neuron, 2 (1989) 1177-1184. 20 Gale, K., Kerasidis, H. and Wrathall, J.R., Spinal cord contusion in the rat: behavioral analysis of functional neurologic impairment, Exp. Neurol., 88 (1985) 123-134. 21 Gibbs, R.B., Chao, M.V. and Pfaff, D.W., Effect of fimbria-fornix and angular bundle transection on expression of the p75NGF mRNA by cells in the medial septum and diagonal band of Broca: correlations with cell survival, synaptic reorganization and sprouting, MoL Brain Res., 11 (1991) 207-219. 22 Goodrich, S.P., Yan, G-C.Y., Bahrenburg, K. and Mansson, P-E., The nucleotide sequence of rat heparin binding growth factor 1 (HBGF-1), Nucl. Acid Res., 17 (1989) 2867. 23 Gospodarowicz, D., Ferrara, N., Schweigerer, L. and Neufeld, G., Structural characterization and biological functions of fibroblast growth factor. Endocr. Rev., 8 (1987) 95-114. 24 Johnson, E.M., Taniuchi, M. and Di Stefano, P., Expression and possible function of nerve growth factor receptor on Schwann cells, Trends Neurosci., 11 (1988) 299-304. 25 Kaplan, D.R., Hempstead, B.L., Martin-Zanca, D., Chao, M.V. and Parada, L.F., The trk protooncogene product: a signal transduction receptor for nerve growth factor, Science, 252 (1991) 554-558. 26 Kerasidis, H., Wrathall, J.R. and Gale, K., Behavioral assessment of functional deficit in rats with contusive spinal cord injury, J. Neurosci. Methods, 20 (1987) 167-189. 27 Klein, R., Jing, S., Nanduri, V., O'Rourke, E. and Barbacid, M., The trk protooncogene encodes a receptor for nerve growth factor, Cell, 65 (1991) 189-197. 28 Knusel, B, Michel, P.P., Schwaber, J.S. and Hefti, F., Selective and nonselective stimulation of central cholinergic and dopaminergic development in vitro by nerve growth factor, basic fibroblast growth factor, epidermal growth factor, insulin and the insulinelike growth factors I and II, J. Neurosci., 10 (1990) 558-570. 29 Koshinaga, M., Sanon, H.R. and Whittemore, S.R., Altered acidic and basic fibroblast growth factor expression following spinal cord injury. Exp. Neurol., 120 (1993) 32-48.

30 Loeb, D.M., Marangos, J., Martin-Zanca, D., Chao, M.V., Parada, L.F. and Greene, L.A., The trk proto-oncogene rescues NGF responsiveness in mutant NGF-nonresponsive PC12 cell line, Cell, 66 (1991) 961-966. 31 Mattson, M.P., Murrain, M., Guthrie, P.B., Kater, S.B. Fibroblast growth factor and glutamate: opposing roles in the generation and degeneration of hippocampal neuroarchitecture, J. Neurosci., 9 (1989) 3728-3732. 32 McNeil, P.L., Muthukrishanan, L., Wader, E., D'Amore, P.A., Growth factors are released by mechanically wounded endhothelial cells, J. Cell Biol., 109 (1989) 811-822. 33 Morrison, R.S., Sharma, A., De Vellis, J., Bradshaw, R.A. Basic fibroblast growth factor supports the survival of cerebral cortical neurons in primary culture, Proc. Natl. Acad. Sci. USA, 83 (1986) 7537-7541. 34 Noble, L.J. and Wrathall, J.R., Spinal cord contusion in rat: morphometric analyses of alteration in the spinal cord, Exp. Neurol., 88 (1985) 135-149. 35 Noble, L.J. and Wrathall, J.R., Correlative analyses of lesion development and functional status after graded spinal cord contusive injuries in the rat, Exp. Neurol., 103 (1989) 34-40. 36 Otto, D., Unsicker, K. and Grothe, C., Basic fibroblast growth factor and nerve growth factor administered in gel foam rescue medial septal neurons after fimbria fornix transection, J. Neurosci. Res., 22 (1988) 83-91. 37 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. 38 Panjabi, M.M and Wrathall, J.R., Biochemical analysis of spinal cord injury and functional loss, Spine, 13 (1989) 1365-1370. 39 Raines, A., Dretchen, K.L., Marx, K. and Wrathall, J.R., Spinal cord contusion in the rat somatosensory evoked potential as function of graded injury, J. Neurotrauma, 5 (1988) 61-70. 40 Reynolds, M.E., Brunello, N., Mocchetti, I. and Wrathall, J.R., Localization of nerve growth factor mRNA in contused rat spinal cord by in situ hybridization, Brain Res., 559 (1991) 149-153. 41 Rifkin, D.B. and Moscatelli, D., Recent development in the cell biology of basic fibroblast growth factor, J. Cell Biol., 109 (1989) 1-6. 42 Riva, M. and Mocchetti, I., Developmental expression of the basic fibroblast growth factor gene in rat brain, Dev. Brain Res., 62 (1991) 45-50. 43 Riva, M.A., Gale, K. and Mocchetti, I., Basic fibroblast growth factor mRNA increases in specific brain regions following convulsive seizures, Mol. Brain Res., 15 (1992) 311-318. 44 Rodriguez-Tebar, A., Dechant, G. and Barde, Y.-A., Binding of brain derived neurotrophic factor to nerve growth factor receptor, Neuron, 4 (1990) 487-492. 45 Schubert, D., Ling, N. and Baird, A., Multiple influences of a Heparin-binding growth factor on neuronal development, J. Cell Biol., 104 (1987) 635-643. 46 Schweigerer, L., Neufeld, G., Friedman, J., Abraham, J.A., Fiddes, J. and Gospodarowicz, D., Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth, Nature, 325 (1987) 257-259. 47 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 FGF and tissue distribution study of its mRNA, Biochem. Biophys. Res. Commun., 157 (1988) 256-263. 48 Squinto, S.P., Stitt, T.N., Aldrich, T.H., Davis, S., Bianco, S.M., Radziejewski, C., Glass, D.J., Masiakowki, P., Furth, M.E., Valenzuela, D.M., DiStefano, P.S. and Yancopoulos, G.D., trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not nerve growth factor, Cell, 65 (1991) 885-893. 49 Tator, C.H., Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms, J. Neurosurg., 75 (1991) 15-26. 50 Tourbah, A., Oliver, L., Jeanny, J.C. and Gumpel, M., Acidic fibroblast growth factor (aFGF) is expressed in the neuronal and glial spinal cord cells of adult mice, Z Neurosci. Res., 29 (1991) 560-568.

51 Walicke, P.A., Basic and acidic fibroblast growth factor have trophic effect on neurons from multiple CNS regions, J. Neurosci., 8 (1988) 2618-2627. 52 Walicke, P., Cowan, W.M., Ueno, N., Baird, A. and Guillemin, R. Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension, Proc. Natl. Acad. Sci. USA, 83 (1986) 3012-3016. 53 Whittemore, S.R., Friedman, P.L., Larhammar, D., Persson, H., Gonzales-Carvajal, M. and Holets, V.R., Rat E-nerve growth factor sequence and site of synthesis in the adult hippocampus. J. Neurosci. Res., 20 (1988) 403-410. 54 Wrathall, J.R., Pettegrew, R.K. and Harvey, F., Spinal cord contusion in the rat: production of graded, reproducible, injury groups, Exp. Neurol., 88 (1985) 108-122.

55 Wrathall, J.R., Bouzoukis, J. and Choiniere, D., Effect of kynurenate on functional deficit from traumatic spinal cord injury, Eur J. Pharmacol., 218 (1992) 273-281. 56 Wrathall, J.R., Teng, Y.D., Choiniere, D. and Mundt, E., Evidence that local non-NMDA receptors contribute to functional deficits in contusive spinal cord injury, Brain Res., 586 (1992) 140-143. 57 Yan, Q., Snider W.D., Pinzone, J.J. and Johnson, E.M., Retrograde transport of nerve growth factor (NGF) in motoneurons of developing rats: assessment of potential neurotrophic effects, Neurons, 1 (1988)335-343.