Methods of inhibiting ECM accumulation in the CNS by inhibition of TGF-.beta.

The present invention relates to methods for preventing, suppressing or treating a CNS pathology characterized by a deleterious accumulation of extracellular matrix in a tissue by contacting the tissue with an agent that inhibits the extracellular matrix producing activity of TGF-.beta.. The methods can be used to prevent, suppress or treat scar formation in the CNS. Agents that are useful in the present methods include neutralizing anti-TGF-.beta. antibodies, Arg-Gly-Asp-containing peptides, decorin and its functional equivalents such as biglycan, and TGF-.beta. antagonists. The present invention further provides methods for preventing, suppressing or treating a CNS pathology characterized by the insufficient accumulation of extracellular matrix. Agents that enhance the production of extracellular matrix, such as TGF-.beta., can be used in such methods. Finally, the present invention provides pharmaceutical compositions containing these agents, which can be administered to patients to inhibit or enhance the production of extracellular matrix in the CNS.

BACKGROUND OF THE INVENTION 
This invention relates generally to growth factors and more specifically, 
to the influence of transforming growth factor-.beta. (TGF-.beta.) on scar 
formation and extracellular matrix production in the central nervous 
system (CNS). 
Complete lesions of neural pathways in the adult mammalian CNS are rarely 
followed by significant functional recovery. After a penetrating injury of 
the brain or spinal cord, a complex sequence of tissue-specific cellular 
events is initiated, including a general inflammatory response, 
angiogenesis, widespread reactive gliosis and the formation of a dense 
permanent scar of mesodermal origin. These responses are accompanied by 
transient neuronal sprouting and synaptogenesis, but in most cases the 
growth responses of neurons are aborted as the glial/meningeal scar 
becomes organized as discussed in Maxwell et al., Phil. Trans. R. Soc. 
Lond. 328:479-499 (1990). 
There are many theories to explain the failure of axonal growth after 
injury to the CNS. They attribute the failure to an absence of trophic 
cues such as growth factors (Logan, Brit. J. Hosp. Med. 43:428-437 (1990)) 
or to the release of growth inhibitory substances (Schnell & Schwab, 
Nature 343:269-272 (1990)). The mature scar, with its dense fibrous 
connective tissue bordered by an astrocytic glia limitans, is a physical 
barrier to axonal growth. It may be that deficiencies in the extracellular 
environment of the growing neurites restrict their growth so that they 
reach the scar tissue after the barrier is formed. Axonal penetration 
through scar tissue does not occur in the CNS. 
Various pathologies are characterized by a deleterious accumulation of 
extracellular matrix materials. For example, in progressive glomerular 
disease, extracellular matrix accumulates in the mesangium or along the 
glomerular basement membrane, eventually causing end-stage disease and 
uremia. Similarly, adult or acute respiratory distress syndrome (ARDS) 
involves the accumulation of matrix materials in the lung, while cirrhosis 
of the liver is characterized by deleterious matrix accumulation evidenced 
by scarring in the liver. 
At present, there are no therapies available to promote successful 
regeneration and functional reconnection of damaged neural pathways. Any 
clinical paradigm designed to promote regeneration of central neural 
pathways must include a regime for reduction of extracellular matrix 
deposition at the wound site. 
Thus, a need exists to determine the factors that regulate accumulation of 
matrix components in the CNS after injury. A need also exists to control 
such factors to prevent, limit or treat pathogenic conditions 
characterized by inappropriate extracellular matrix formation in the CNS. 
The present invention satisfies these needs and provides related 
advantages as well. 
SUMMARY OF THE INVENTION 
The present invention relates to methods for preventing, suppressing or 
treating a CNS pathology characterized by a deleterious accumulation of 
extracellular matrix in a tissue by contacting the tissue with an agent 
that inhibits the extracellular matrix producing activity of TGF-.beta.. 
The methods can be used to prevent, suppress or treat scar formation in 
the CNS. 
Agents that are useful in the present methods include, for example, 
neutralizing anti-TGF-.beta. antibodies, Arg-Gly-Asp-containing peptides, 
decorin and its functional equivalents such as biglycan. Additionally, 
such agents can also be TGF-.beta. antagonists that compete with 
TGF-.beta. in binding to a TGF-.beta. receptor. Pharmaceutical 
compositions containing these agents can be administered to the patients 
to inhibit the activity of TGF-.beta.1 in the CNS. 
The present invention further relates to methods for preventing, 
suppressing or treating a CNS pathology characterized by an insufficient 
accumulation of extracellular matrix by contacting a tissue with an agent 
that promotes extracellular matrix formation. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention generally relates to CNS injuries, and more 
particularly the presence of TGF-.beta.1 in injured CNS tissues. Although 
mRNA for TGF-.beta.1, TGF-.beta.2 and TGF-.beta.3 are detectable in 
embryonic mouse brain, only TGF-.beta.2 and TGF-.beta.3 have been 
localized in unlesioned adult rat brain. In the experiments described 
below, very low levels of TGF-.beta.1 expression were detected in the 
unlesioned adult rat brain both by in situ hybridization and 
immunostaining. This observation is in agreement with the results 
described in Miller et al., Molec. Endocrinol. 3:1926-1934 (1989) and 
Wilcox & Derynck, Mol. Cell. Biol. 8:3415-3422 (1988), in which 
TGF-.beta.1 expression was not detected in adult mouse brain. 
A number of growth factors are known to mediate various injury responses in 
peripheral tissues. In particular, TGF-.beta. is a potent stimulator of 
extracellular matrix deposition in peripheral tissue wounds. TGF-.beta. 
has a profound influence on extracellular matrix production, including 
increasing collagen, fibronectin, and proteoglycan expression. This growth 
factor also increases integrin expression, decreases the synthesis of 
proteases which degrade extracellular matrix components such as 
collagenase and transin, and increases the expression of protease 
inhibitors, such as the plasminogen activator inhibitor type 1 and the 
tissue specific inhibitor of metalloprotease. 
TGF-.beta. is a multifunctional cytokine that plays an important role in 
regulating repair and regeneration following tissue injury. Three isoforms 
of TGF-.beta., TGF-.beta.1, 2, and 3, are expressed in mammals and to date 
show similar properties in vitro. Platelets contain high concentrations of 
TGF-.beta., and upon degranulation at a site of injury, release TGF-.beta. 
into the surrounding tissue. TGF-.beta. then initiates a sequence of 
events that promotes healing including (1) chemoattraction of monocytes, 
neutrophils, and fibroblasts, (2) autoinduction of TGF-.beta. production 
and stimulation of monocytes to secrete interleukin-1 (IL-1), tumor 
necrosis factor and other cytokines, (3) induction of angiogenesis and 
cell proliferation, (4) control of inflammation and cell toxicity by 
acting as a potent immunosuppressant and inhibitor of peroxide release, 
and (5) increased deposition of extracellular matrix. 
The effect of TGF-.beta. on extracellular matrix is a key feature of its 
functional activities. TGF-.beta. stimulates the synthesis of individual 
matrix components such as fibronectin, collagens and proteoglycans and 
simultaneously blocks matrix degradation by decreasing the synthesis of 
proteases and increasing the levels of protease inhibitors as described in 
Edwards et al., EMBO 6:1899 (1987) and Laiho et al., J. Biol. Chem. 
262:17467 (1987). TGF-.beta. also increases the expression of integrins 
and changes their relative proportions on the surface of cells in a manner 
that could facilitate adhesion to matrix as reported in Ignotz & Massague, 
Cell 51:189 (1987). 
The mature form of TGF-.beta. is comprised of two identical chains, each of 
112 amino acids. The amino acid sequence of TGF-.beta. is as follows: 
Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys Val Arg Gln 
Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys 
Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp 
Thr Gln Tyr Ser Lys Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser 
Ala Ala Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr 
Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser Cys 
Lys Cys Ser. (SEQ. ID. NO. 1) 
In peripheral tissues TGF-.beta. is a potent stimulant for scar tissue 
formation characterized by the excessive accumulation of extracellular 
matrix components. Various agents have been found that block the 
stimulatory effect of TGF-.beta. on proteoglycan production resulting in 
reduced extracellular matrix formation in peripheral tissues as described, 
for example, in Ruoslahti and Yamaguchi, Cell 64:867-869 (1991). Such 
agents include, for example, anti-TGF-.beta. antibodies, RGD-containing 
peptides and decorin. 
However, the role of TGF-.beta.1 in CNS wounds was not known until the 
discovery relating to the present invention. The focal elevation of 
TGF-.beta.1 mRNA and protein observed three days after lesion, suggests 
that this growth factor plays a role in regulating the CNS responses to 
injury. The association of TGF-.beta.1 mRNA and protein with 
injury-responsive cells such as glia, neurons and vascular endothelial 
cells, indicates that the factor is endogenously produced, and not simply 
supplied to the wound via blood platelets. The observations made in this 
model agree with those obtained by others who have also demonstrated 
increased TGF-.beta.1 mRNA in rat hippocampus after entorhinal cortex 
lesion by northern blot hybridization (Nicols et al., Neurosci. Res. 
28:134-139 (1991)) and by in situ hybridization in rat cerebral cortex, 
striatum and hippocampus after a hypoxic-ischemic insult (Klempt et al., 
Proc. 73rd Ann. Meeting Am. Endocrin. Soc. p. 1809 (Abstract 1991)). The 
results are particularly surprising because the presence of TGF-.beta.1 in 
injured CNS tissue was believed to be a result of a compromised blood 
brain barrier. However, in the studies described below, it has been shown 
that the blood brain barrier is regenerated before scarring occurs. 
The in vivo infusion studies detailed in the examples below further 
evidence that TGF-.beta.1 is a prime regulator of matrix production in the 
CNS after injury. A dramatic increase in fibrous matrix material deposited 
within the wound is observed in all animals infused with recombinant 
TGF-.beta.1. Thus, in the injured CNS, as in peripheral tissues, 
TGF-.beta.1 enhances matrix deposition and therefore promotes fibrous scar 
formation. The reactive gliosis that occurs in the neuropile surrounding 
the wound is apparently unaffected by the treatment as is astrocyte 
association into a glial membrane, suggesting that, for this part of the 
response at least, TGF-.beta.1 is not a limiting trophic factor. The 
observed dramatic increase in number of cells of the macrophage/microglial 
lineage in the injured neuropile of TGF-.beta.1 infused animals suggests 
an additional role in injury for TGF-.beta.1, as a chemoattractant for 
blood-derived cells that themselves produce multiple trophic factors. 
That TGF-.beta.1 is produced by cells in the damaged tissue suggests that 
it is acting as an endogenous stimulant of scarring responses in the CNS 
and, in particular, of matrix deposition. Immunoneutralization of 
endogenous TGF-.beta.1 activity results, in some cases, in a dramatic 
reduction in matrix deposition in the CNS wound, thus establishing an 
intrinsic activity for endogenous TGF-.beta.1. The results suggest that a 
reduction of the dense permanent scar that is deposited at the site of 
injury, and which blocks the path of regenerating neurons, is one step 
towards achieving functional reconnection of damaged neural pathways to 
their target organs. 
The reduction in macrophage/microglial cell number in the immunoneutralized 
wound and the lack of an organized glial limiting membrane, despite the 
presence of a clear reactive gliosis response, again suggests that 
TGF-.beta.1 is exerting multiple effects in these damaged tissues. Thus, 
endogenous TGF-.beta.1 may act as a chemoattractant for blood-derived 
cells and for activated astrocytes, as well as a potent desmoplastic 
agent, promoting matrix deposition by the invading fibroblasts. The 
functional consequences of the immunoneutralizing effects reported here 
are at present unknown, but in this study we could see no evidence of 
active nerve regeneration (assessed using GAP 43 and RT 97 antibodies as 
markers of regenerating axons) in any of the treated animals after 
fourteen days. Presumably, additional neurotrophic agents may be required 
to sustain this aspect of the injury response. 
In this study, although the response to infused recombinant TGF-.beta.1 was 
consistent in all animals, the response to TGF-.beta.1 
immunoneutralization was consistently more variable. This variability may 
be attributed to the effective concentration and bioactivity of the 
infused antibody within the target tissue and to potential compensatory 
mechanisms of other trophic factors which may be contributing to the 
regulation of the cellular responses examined. 
The infusion experiments described below are the first to directly address 
the question of whether scar production in the CNS is amenable to 
modulation in vivo. While the modification of the injury response has been 
achieved using TGF-.beta.1-related molecules, immunoneutralization of 
TGF-.beta.1 in this model suggests that the course of scar production can 
be changed, particularly reduced or prevented. The development of novel 
therapies based on the manipulation of growth factor bioavailability 
within the wound in the acute phase of the injury response is a novel 
strategy. 
The results of the studies relating to the present invention clearly 
implicate TGF-.beta.1 as a regulator of scar production after a 
penetrating injury to the brain or spinal cord. Furthermore, because this 
scar formation can preclude neuronal recovery, the results indicate a 
potential use for TGF-.beta.1 antagonists as an adjunct to those therapies 
designed to promote regeneration and reconnection of damaged neural 
pathways. 
Accordingly, the present invention provides a method for preventing, 
suppressing or treating CNS pathologies characterized by a deleterious 
accumulation of extracellular matrix in a tissue. Such methods can be 
accomplished by contacting the tissue with an agent that inhibits the 
extracellular matrix producing activity of TGF-.beta.1. The agent can be, 
for example, a neutralizing anti-TGF-.beta. antibody or a functional 
fragment, or an Arg-Gly-Asp-containing peptide. Preferably, such an 
Arg-Gly-Asp-containing peptide is between 4 and 50 amino acids in length. 
Additionally, agents that act as TGF-.beta. antagonists, such as fragments 
of TGF-.beta. having sequences that bind to a TGF-.beta. receptor, can be 
used in the present methods. Such antagonists'should not induce the 
production of extracellular matrix, but will competitively bind to such 
receptors to prevent TGF-.beta. binding. 
The agent can also be decorin or its functional equivalent. As used herein, 
"decorin" refers to a proteoglycan having substantially the structural 
characteristics attributed to it in Krusius and Ruoslahti, PNAS (USA) 
83:7638 (1986). Human fibroblast decorin has substantially the amino acid 
sequence presented in Krusius and Ruoslahti, supra. "Decorin" refers both 
to the native composition and to modifications thereof which substantially 
retain the functional characteristics. Decorin core protein refers to 
decorin that no longer is substantially substituted with glycosaminoglycan 
and is included in the definition of decorin. Decorin can be rendered 
glycosaminoglycan-free by enzymatic treatment, mutation or other means, 
such as by producing recombinant decorin in cells incapable of attaching 
glycosaminoglycan chains to a core protein or by synthesizing the core 
protein, all by means well known in the art. 
Functional equivalents of decorin include modifications of decorin that 
retain its functional characteristics and molecules that are homologous to 
decorin, such as biglycan and fibromodulin, for example, that have the 
similar functional activity of decorin. Modifications can include, for 
example, the addition of one or more side chains that do not interfere 
with the functional activity of the decorin core protein. 
The agents useful in the methods can be obtained by purifying the native 
protein or by proteolytic digestion of such proteins to obtain 
functionally active fragments according to methods known in the art. 
Alternatively, such agents can be synthesized or produced recombinantly by 
methods known in the art. 
The present invention further provides methods for preventing, suppressing 
or treating a CNS pathology characterized by the insufficient accumulation 
of extracellular matrix components to promote scar formation. These 
methods can be accomplished by administering TGF-.beta. or a functional 
fragment that promotes extracellular matrix production to a patient in 
need of such therapy. Such methods can be used when inadequate scar 
formation can or does result in a CNS pathology. 
Pharmaceutical compositions containing agents that inhibit the activity of 
TGF-.beta. or increase the concentration of TGF-.beta. and a 
pharmaceutically acceptable carrier can be administered to a patient to 
prevent, enhance or otherwise treat CNS scar formation. Suitable 
pharmaceutically acceptable carriers include, for example, hyaluronic acid 
and aqueous solutions such as bicarbonate buffers, phosphate buffers, 
Ringer's solution and physiological saline supplemented with 5% dextrose 
or human serum albumin, if desired. Other pharmaceutical carriers known to 
those skilled in the art are also contemplated. The pharmaceutical 
compositions can also include other reagents that are useful for the 
prevention or treatment of the various CNS pathologies characterized or 
associated with the accumulation of extracellular matrix. The dosage of 
such pharmaceutical compositions can be readily determined by those 
skilled in the art based on various factors such as, for example, the type 
and extent of the injury, the age of the patient and the agent used. 
The present invention further relates to methods of detecting the presence 
of various CNS pathologies of a tissue characterized by an excessive 
accumulation of extracellular matrix components by determining the level 
of TGF-.beta.1 in the tissue. Such detection methods can be accomplished 
by the procedures described in the examples below or by other methods 
known in the art.

The following examples are intended to illustrate but not limit the 
invention. 
EXAMPLE I 
Materials 
Unless specified, all reagents were analytical grade from Sigma Chemical 
Co. Ltd. (St. Louis, Mo.) or Poole (United Kingdom). Radioisotopes were 
obtained from Amersham International (Arlington Heights, Ill.). 
Recombinant human TGF-.beta.1 was obtained from R and D Systems 
(Minneapolis, Minn.). Two anti-TGF-.beta.1 antibodies were used in this 
study. The inactivating antibody used in the infusion experiments was 
raised in the turkey against intact native human TGF-.beta.1. A different 
antibody was used for histochemistry, which was raised in the rabbit 
against amino acids 1-30 of human TGF-.beta.1. Both of the 
anti-TGF-.beta.1 antibodies were prepared and characterized by M. B. Sporn 
and K. C. Flanders (National Institutes of Health, Bethesda, Md.) and 
described in Logan et al., supra. 
EXAMPLE II 
Animals and Surgery 
Surgical and animal care 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. 
Groups of adult, female Sprague-Dawley rats (250 grams) were used in the 
study. Animals were anaesthetized with an intraperitoneal injection of a 
mixture of acepromazine (1.875 mg/kg), ketamine (3.75 mg/kg) and xylazine 
(1.9 mg/kg). Following craniotomy, a stereotactically defined, 4 mm deep, 
rostro-caudal knife-wound incision was made vertically into the right 
occipital cortex, corpus callosum and presubiculum, placed 1 mm anterior 
to Bregma/1.4 mm lateral of the mid-line, so it penetrated the anterior 
lateral ventricle at some point in its length. Control animals underwent 
craniotomy but no lesion was made. 
Three days after surgery, four lesioned and two control rats were put under 
deep anaesthesia with the same anaesthetic and perfused transcardially 
with 300 ml of 0.9% (wt/vol) saline, 250 ml of 4% (wt/vol) 
paraformaldehyde (PFA) in 0.1 M acetate buffer, pH 6.5, followed by 500 ml 
of 4% (wt/vol) PFA plus 0.05% (wt/vol) glutaraldehyde in 0.1 M borate 
buffer, pH 9.5, using the pH shift method described in Simmons et al., J. 
Histotechnology 12:169-181 (1989), incorporated herein by reference. The 
brains were removed and post-fixed overnight at 4.degree. C. in 4% 
(wt/vol) PFA in 0.1 M borate buffer containing 10% (wt/vol) sucrose. These 
brains were processed for in situ hybridization and immunoperoxidase 
staining to reveal expression of TGF-.beta.1 mRNA and protein in the 
tissue surrounding the lesion. 
The remaining three groups of six animals underwent a further surgical 
procedure at the same time as the placement of the lesion. In these 
animals, a vertical stainless steel cannula was inserted through the 
cranium into the posterior of the right lateral ventricle. The cannula was 
cemented into place with a dental cement platform which was stabilized by 
three stainless steel machine screws inserted into the cranium distal to 
the site of cannulation. The cannula was attached under the skin, via a 
flexible vinyl catheter, to a ready-primed Alzet mini-osmotic pump (model 
2002, Alza Corporation, Palo Alto, Calif.) which was inserted into a 
sub-cutaneous pouch made in the dorsal neck region of the animal. The 
pumps supplied test agents to the cannula at a prescribed rate (0.5 
.mu.l/hr) and dose over a 14 day period. 
In the main experiment the mini-osmotic pumps were primed to supply a basic 
infusion vehicle of phosphate-buffered artificial cerebrospinal fluid: 150 
mM NaCl, 1.8 mM CaCl.sub.2, 1.2 mM MgSO.sub.4, 2.0 mM K.sub.2 HPO.sub.4, 
10.0 mM glucose, pH 7.4. In addition, 0.1% autologous rat serum was 
included in the vehicle to reduce absorption losses within the infusion 
apparatus. TGF-.beta.1 protein and anti-TGF-.beta.1 antiserum were diluted 
to their appropriate concentrations directly into this vehicle. In vitro 
experiments to monitor the stability of the infused TGF-.beta.1 and 
anti-TGF-.beta.1 antibody indicated that biological activity of both 
protein and antibody is preserved at 37.degree. C. over a 14 day period. 
Six animals were infused with vehicle containing a 1:100 dilution of 
non-immune turkey serum (controls), six animals with vehicle containing a 
1:100 dilution of turkey anti-TGF-.beta.1 antiserum raised in turkeys 
against native human TGF-.beta.1, and six animals with vehicle containing 
150 ng/ml recombinant human TGF-.beta.1. Thus, the TGF-.beta.1-infused 
animals received 1.8 ng/day recombinant human TGF-.beta.1 and the 
antiserum-infused animals received 0.12 .mu.l/day non-immune or 
anti-TGF-.beta.1 turkey antiserum. Fourteen days after cannula 
implantation and continuous delivery of experimental infusion solutions, 
all animals were processed as previously described and histochemical 
evaluation of the lesion site was performed by immunofluorescent staining. 
EXAMPLE III 
In Situ Hybridization of TGF-.beta.1 mRNA 
In situ hybridization of TGF-.beta.1 mRNA used the Hind III-Xba I fragment 
of 0.985 kBp, derived from the major coding region of the rat TGF-.beta.1 
precursor (Qian et al., Nucl. Acids Res. 18:3059 (1990)), which was 
sub-cloned into pBluescript SK+ (Stratagene, San Diego, Calif.). The 
antisense RNA strand of the coding sequence was transcribed using T7 
polymerase and .sup.35 S-UTP according to manufacturer's instructions. 
.sup.35 S-UTP labelled RNA probes encoding sense strands of 5' non-coding 
sequences were prepared with T3 RNA polymerase and used for alternate 
control tissue sections. 
The fixed brains were frozen in O.C.T. compound (Miles Laboratories Inc., 
Napeville, Ill.) in dry ice and stored at -80.degree. C. At a later date, 
20 .mu.m frozen sections were cut and collected in cryoprotectant solution 
(50% 0.05 M sodium phosphate buffer, pH 7.3, 30% ethylene glycol and 20% 
glycerol) and stored at -20.degree. C. Subsequently cryoprotected brain 
sections were washed thoroughly in phosphate-buffered saline (PBS), 
mounted on poly-L-lysine coated slides, dried under vacuum and stored at 
-80.degree. C. until use. 
For analysis, sections were digested with 10 .mu.g/ml of proteinase K in 
0.1 M Tris (pH 8.0) containing 50 mM EDTA at 37.degree. C. for 30 minutes 
and then rinsed in deionized water, followed by an incubation in 0.1 M 
triethanolamine hydrochloride (TEA), pH 8.0 for 3 minutes. Sections were 
acetylated for 10 minutes with 0.25% (wt/vol) acetic anhydride in 0.1 M 
TEA, rinsed in 2.times.SSC (prepared from a 20.times.stock solution which 
contains 3 M NaCl and 0.015 M sodium citrate), dehydrated through a graded 
series of ethanol washes and then air dried for 2 hours before 
hybridization. 
Hybridization with the labelled TGF-.beta.1 antisense or sense probes 
(1.times.10.sup.7 cpm/ml) was performed at 55.degree. C. overnight in 10 
mM Tris (pH 8.0) containing 50% (wt/vol) formamide, 0.3 M NaCl, 1 mM EDTA, 
10 mM dithiothreitol (DTT), 1.times.Denhardt's solution (0.1 g Ficoll 400, 
0.1 g polyvinylpyrrolidone, 0.1 g of bovine serum albumin), and 10% 
(wt/vol) dextran sulfate. After hybridization, sections were rinsed for 1 
hour in 4.times.SSC and treated with 25 .mu.g/ml of ribonuclease A in 10 
mM Tris (pH 8.0) containing 0.5 M NaCl, 1 mM EDTA at 37.degree. C. for 30 
minutes. This treatment was followed by increasing high stringency washes 
of SSC containing 1 mM DTT, followed by a final wash in 0.1.times.SSC at 
65.degree. C. for 30 minutes. 
Slides were then dehydrated through a graded series of ethanol, until 
absolute ethanol, dried under vacuum and exposed to .beta.max hyperfilm 
(Amersham) for 5 days at 4.degree. C. to examine gross changes in mRNA. 
For microscopic analysis, slides were coated with Kodak NTB-2 liquid 
autoradiograph emulsion and exposed at 4.degree. C. for 2-3 weeks. They 
were developed in Kodak D-19, rinsed briefly in water, and fixed in Kodak 
rapid fixer. After washing in distilled water for at least 45 minutes, 
slides were counterstained with Harris' haematoxylin in order to visualize 
the cells. Silver grains were examined by dark field and bright field 
microscopy. 
Bright and dark field views of the lesion site were taken. After three 
days, an intense signal was observed for TGF-.beta.1 mRNA in the neuropile 
bordering the lesion site. This focal elevation of TGF-.beta.1 mRNA 
suggests a local expression of TGF-.beta.1 within the damaged neural 
tissue in response to injury. Under higher power, the bright field view 
revealed that the signal was mainly associated with cells of neuronal and 
astrocytic visual phenotype, although signal was also seen associated with 
endothelial cells of the microvasculature and in the local meninges. The 
increased level of signal seen in the lesioned hemisphere was striking 
when compared to that seen in the contralateral hemisphere or in sections 
of unlesioned brain, which were processed identically and simultaneously. 
Since the signal observed in the brains of control animals was also 
minimal, it seems that there is normally very low expression of 
TGF-.beta.1 mRNA in this tissue. The hybridization signal observed in 
sections from lesioned rat brains was specific, since adjacent tissue 
sections hybridized with the sense strand of cRNA show no signal. 
EXAMPLE IV 
Immunoperoxidase Staining of TGF-.beta.1 
Immunoperoxidase staining for TGF-.beta.1 in 20 .mu.m frozen sections of 
brain was accomplished using the ABC Vactastain Elite kit (Vector 
Laboratories Ltd., Burlingame, Calif.) according to the manufacturer's 
instructions. The primary antiserum raised against TGF-.beta.1 has been 
previously described and characterized in Flanders et al., J. Cell Biol. 
108:653-660 (1989). It is an IgG fraction of a rabbit polyclonal, raised 
against amino acids 1-30 of human TGF-.beta.1, which was purified by 
passage over a protein A-Sepharose column. 
For analysis, the cryoprotected 20 .mu.m sections were washed thoroughly in 
PBS, mounted onto gelatin coated slides, rinsed again in PBS and the 
endogenous peroxidase was quenched by incubating the sections in 0.3% 
(vol/vol) hydrogen peroxide in PBS for 30 minutes. The sections were 
rinsed in PBS and incubated for 30 minutes in 1.5% (vol/vol) normal goat 
serum, diluted in PBS containing 0.3% (vol/vol) Triton X-100, in order to 
block non-specific binding. Following this procedure, the sections were 
incubated for 24 hours at 4.degree. C. with protein-A purified rabbit 
anti-TGF-.beta.1 antibody (0.015 mg/ml) diluted in PBS containing 0.3% 
(vol/vol) Triton X-100 and 1% (wt/vol) bovine serum albumin (BSA). They 
were then rinsed and incubated with a 1:200 dilution of biotinylated goat 
anti-rabbit IgG (Vector) for 45 minutes, rinsed and incubated with 
avidin-biotin-peroxidase complex (Vector) for 30 minutes. After rinsing in 
PBS, the sections were treated with 0.5 mg/ml of 3'3'-diaminobenzidine 
(DAB), diluted in PBS containing 0.01% (vol/vol) hydrogen peroxide for 5 
minutes. All steps were separated by buffer washes consisting of PBS, pH 
7.4, containing 0.3% (vol/vol) Triton X-100. The sections were finally 
washed in PBS, counterstained with Harris' haematoxylin, dehydrated, 
cleared, and mounted. Sections incubated with anti-TGF-.beta.1 antibody 
pre-incubated with recombinant TGF-.beta.1 or without primary antibody 
were used as controls. Sections processed with the control procedures 
failed to stain. 
The immunocytochemical localization of TGF-.beta.1 in the wound was 
determined three days after injury. The appearance of immunoreactive 
TGF-.beta.1 seen in damaged neural tissue correlates with the extent of 
mRNA induction observed by in situ hybridization. Under high 
magnification, the predominant cell types (by morphological criteria) 
localizing strong TGF-.beta.1 immunoreactivity three days after injury 
were the astrocyte and macrophage. The staining is mostly limited to 
damaged neural tissues bordering the forming glia limitans and thus 
appears to be primarily extracellular. In this study, no immunoreactivity 
was observed in the contralateral hemisphere or in sections of normal, 
unlesioned brain processed simultaneously. 
EXAMPLE V 
Immunofluorescent Staining of the Wound 
After perfusion fixation, the brains of the fourteen day cannulated and 
continuously infused animals were washed in PBS overnight at 4.degree. C., 
dehydrated in graded alcohols, embedded in polyester wax (melting point 
37.degree. C.) and stored at 4.degree. C. Sections (7 .mu.m) were cut on a 
microtome fitted with a cooled chuck and floated onto a gelatin solution 
(10 mg/ml) on subbed slides and air-dried. 
The antibodies used to identify cellular changes in the wounds were rabbit 
anti-bovine glial fibrillar acidic protein (GFAP) as a marker of activated 
astrocytes, rabbit anti-mouse fibronectin to visualize matrix deposition 
within the wound and rabbit anti-mouse ED1 as a marker of cells of the 
macrophage/microglial lineage. All of these antibodies were obtained from 
Dakopatt Ltd. (High Wycombe, U.K.) and were used at a dilution of 1:200 in 
PBS containing 1% (wt/vol) BSA. 
The mounted brain sections were dewaxed, rehydrated and placed in PBS 
containing 0.1% (vol/vol) Tween-20 for 15 minutes. They were then 
incubated in the specific antibody for 1-12 hours at room temperature. 
After three washes in PBS, sections were incubated in goat anti-rabbit IgG 
conjugated with fluorescein isothiocyanate (FITC, Sigma), diluted to 
1:100. The sections were washed in three changes of PBS and mounted in a 
non-quenching mountant. For controls, either the first or second antibody 
was omitted, all were negative. The sections were examined with an Olympus 
BH-2 microscope with a fluorescent attachment. Photomicrographs were taken 
on Ilford HP5 film, rated at 400 ISO. 
EXAMPLE VI 
General Appearance of the Lesion 
Three days after the induced injury, oedema in the wound was still 
extensive. Many macrophages were present in the central lumen of the wound 
but macrophages and microglia were also seen in the damaged neuropile. 
Numerous meningeal-fibroblasts were present in the center of the wound in 
the superficial cortex and scar tissue was beginning to form between the 
cut edges of the cortical neuropile, but had not yet penetrated the depths 
of the lesion. Reactive astrocytes were particularly numerous at the 
damaged margin of the neuropile, and also extended well into the intact 
neuropile. 
At 14 days the major cellular events are complete. Briefly, the scar tissue 
had contracted, bringing the cut borders of the neuropile close together. 
Some residual macrophages were visible in the fibrous tissue which had 
been deposited at the center of the lesion site. A fully developed glia 
limitans was present that lines the cut margins of the neuropile and 
surrounds a thin core of fibrous scar tissue. Reactive astrocytes were 
still visible through the damaged and intact neuropile, but the gliosis 
was receding. 
EXAMPLE VIII 
Characterization of the Wound After Infusion of Recombinant TGF-.beta.1 and 
TGF-.beta.1 Neutralizing Antibodies 
The efficacy of the infusion method for the delivery of test agents to 
cells at the site of cannulation and at the site of lesion was tested by 
infusing a 1:100 dilution of non-immune rabbit antiserum into the right 
lateral ventricle of two lesioned rats over a two week period (0.12 .mu.l 
antiserum/day directly into the CSF). After fixation by perfusion, the 
brains were processed for immunoperoxidase staining using an anti-rabbit 
IgG antibody (Dakopatt Ltd., High Wycombe, U.K.), to examine the extent of 
penetration of the infused antiserum. The results of this test show an 
extensive distribution of the infusate is revealed throughout the tissue 
surrounding both the site of cannulation and the site of lesion. 
Penetration of infused recombinant protein and antiserum into the tissue 
surrounding the lesion site was confirmed at fourteen days in each 
experimental animal by immunoperoxidase staining. By this time the 
endogenous expression of TGF-.beta.1 has virtually subsided and negligible 
TGF-.beta.1 is detectable in the control, vehicle-infused animals. There 
was no observed effect of any of the continuous infusions into the CSF on 
the gross morphology of the ventricles, assessed by visual and microscopic 
inspection of tissue sections. The effect on the injury response of 
modulating TGF-.beta.1 availability at the wound site by continuous 
infusion of TGF-.beta.1-related molecules is discussed in more detail 
below. Briefly, the TGF-.beta.1 infused wounds show a normal reactive 
gliosis response, an abnormal amount of fibronectin deposition and an 
increased number of macrophage and microglial cells when compared to 
control. The anti-TGF-.beta.1 antiserum infused animals show a normal 
reactive gliosis response, but no organization of astrocytes to form a 
limiting glia limitans at the margins of the damaged neuropile. There is 
also a complete absence of immunoreactive fibronectin within the wound and 
a reduced number of macrophage/microglial cells when compared to control. 
A. Control, Vehicle Infusions 
In rats with wounds that had been continuously infused with vehicle plus 
non-immune turkey IgG, immunofluorescent staining of sections after 14 
days shows the scar tissue has contracted bringing together the cut 
borders of the neuropile. In the center of the wound was a thin layer of 
fibrous tissue represented by the fluorescent fibronectin, which is 
visible after staining with anti-fibronectin antibody. Anti-ED1 antibody, 
which detects cells of the macrophage/microglial lineage, revealed 
residual cells in the center of the wound and in neural tissues bordering 
the scar. GFAP-positive astrocytes were still abundant in the tissue 
around the wound and are particularly numerous at the lesion edge, where 
they associate to construct a glia limitans which helps to reform the 
blood-brain barrier. The scar of these animals was deemed to be normal 
when compared to scars seen in lesioned, non-infused rats. 
B. Recombinant TGF-.beta.1 Infusions 
Continuous infusion of the wound with 1.8 ng/day recombinant human 
TGF-.beta.1 resulted in a clear enhancement of scarring with matrix 
deposition being markedly increased compared to lesioned, non-infused 
rats. The increase in matrix deposition was evidenced by the presence of 
an abnormally large area of immunoreactive fibronectin in the center of 
the wound, which resulted in a wide separation of the normally closely 
apposed cut faces of the neuropile. The fibronectin deposition was 
accompanied by an exactly coincident increase in collagen IV and laminin 
deposition. TGF-.beta.1 treatment also dramatically increased the number 
of residual macrophage/microglia cells in the neuropile, which was 
detected by using anti-ED 1 antibody, but had no apparent effect on the 
extent of reactive gliosis observed, which was visualized with anti-GFAP 
antibody. The continuous glia limitans, formed by the reactive astrocytes 
and marking the borders of the cut neuropile, was evident in these wounds. 
C. Anti-TGF-.beta.1 Antiserum Infusions 
Immunoneutralization of endogenous TGF-.beta.1 with 0.12 .mu.l/day of 
turkey anti-TGF-.beta.1 antiserum confirmed the effects of exogenous 
TGF-.beta.1 observed above. In direct contrast, this treatment markedly 
reduced the amount of fibrous scar tissue deposited in the wound. In this 
experiment, the extent of scar reduction was variable, four out of six 
rats responding to the treatment, with two of these showing an almost 
complete absence of matrix deposition in the wound. One such animal showed 
no apparent fibronectin deposition in the wound after fourteen days, as 
indicated by the absence of fibronectin immunoreactivity and a reduced 
residual number of ED 1-expressing macrophages/microglia. The neural 
tissue around this wound contained numerous reactive GFAP-positive 
astrocytes. However, these have not become organized into a limiting glial 
membrane at the margin of the lesion. 
Although the invention has been described with reference to various 
embodiments, it should be understood that various modifications can be 
made without departing from the spirit or scope of the invention. 
Accordingly, the invention is limited only by the following claims. 
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# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 1 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 112 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Th - #r Glu Lys Asn Cys Cys 
# 15 
- Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys As - #p Leu Gly Trp Lys Trp 
# 30 
- Ile His Glu Pro Lys Gly Tyr His Ala Asn Ph - #e Cys Leu Gly Pro Cys 
# 45 
- Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr Se - #r Lys Val Leu Ala Leu 
# 60 
- Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Al - #a Pro Cys Cys Val Pro 
#80 
- Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr Ty - #r Val Gly Arg Lys Pro 
# 95 
- Lys Val Glu Gln Leu Ser Asn Met Ile Val Ar - #g Ser Cys Lys Cys Ser 
# 110 
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