USE OP NGF FOR THE PREPARATION OF MEDICAMENTS FOR THE CURE OF REACTIVE GLIOSIS

DESCRIPTION

The present invention relates to the use of NGF for the preparation of a medicament for the cure and/or prevention of reactive gliosis and therapeutic methods for the cure and/or prevention of reactive gliosis by NGF administration.

STATE OF THE PRIOR ART Astrocytes constitute one of the main components of the glia, and are the most abundant cells in the Central Nervous System (CNS) ; their numeric ratio with respect to nervous cells increases with the increasing complexity of the CNS. For about a century glial cells have received scarce attention in the neuroscience field. Studies conducted in the last decade have highlighted a key role of these elements within the scope of both physiological and pathological processes. Glial cells, moreover, both within the developing nervous system and in the mature one, play a central role in synapse formation, maintenance and function.

In the adult brain, astrocytes participate in the propagation of the nervous impulse by releasing transmitters (gliotransmission) such as glutamate, ADP, D-serine and eicosanoids, able to modulate synaptic activity. Moreover, by releasing such substances astrocytes exert different effects on neighbouring neurons, on the same glia and on blood vessels. In fact, glial cells express a pool of receptors, transporters and ionic channels, by which they can receive and integrate neuronal messages and modulate the efficacy of synapses. Therefore, it can be stated that glia actively participates to brain function. For a long time, astrocytes have erroneously been considered as support cells. It has recently been recognized that astrocytes are organized in separate territories, yet forming a

syncytium, and that they are communication elements within the brain. Therefore, they can generate various regulatory signals and have an interface and connection function between neurons and between neurons and vessels. These findings have led to the concept of the "tripartite" synapse. This type of contact occupies a privileged position in the central nervous system at the interface between capillaries and neurons. Involved in cerebral metabolism, astrocytes may, therefore, account for the modifications of the cerebral blood flow, an indirect index of the cortical activity which is recorded by functional imaging techniques (fMRI) .

Numerous data prove that astrocytes play a specific and essential role not only in normal cerebral functions, but also in the development and progression of neurodegenerative processes. Astrocytes are arranged in an, orderly way and each cell covers a well-delimited territory, located so as to allow local interactions. Astrocytes interface with the microvasculature and might contact several neurons, many nerve fibers and hundreds to thousands of synapses. Following an injury or insult to the CNS, it is determined a glial response known as reactive gliosis, characterized by cellular hypertrophy, increase in the expression of cytoskeleton proteins such as vimentin and GFAP (glial fibrillary acidic protein) , upregulation of intermediate filament (IF) proteins, reexpression of nestin and, in the first week, also cellular division (Okada, S. et al, 2006), and upregulation of factors participating in the formation of the IF network. It is reported in the literature (Bushong et al 2002, 2004) that in reactive gliosis the IF network becomes very prominent, in particular in the main processes and the soma of astrocytes, and this is accompanied by altered expression of many proteins (Eddleston and Mucke 1993, Hernandez et al 2002) .

Therefore, the cells undergo morphologic and metabolic changes. The process of astrocyte activation is

to date rather enigmatic but is now regarded as a reaction of the glia, with specific functional and structural characteristics. Activated astrocytes are present in various pathological conditions of the central nervous system, such as stroke, trauma, growth of a tumor, or neurodegenerative disease ^' such as Alzheimer' s disease, Parkinson's disease, triplet diseases, amyotrophic lateral sclerosis, multiple sclerosis.

Gliosis, therefore, according to the region in which it onsets, would seem to be at the basis of the development of many pathologies: from neurodegenerative disorders (cerebellar gliosis in Spinocerebellar Ataxia type 1 - SCAl -, mesencephalic-striatal gliosis in Parkinson' s disease, cortical gliosis in Alzheimer' s disease) , to neuropathic pain. Hypertrophic and activated glial cells can thus determine a sort of compression of the surrounding parenchyma by inducing a suffering thereof and, concomitantly to the disregulation of the above-indicated proteins, by actively contributing to create a substrate for the development of pathologies.

Gliosis, beside fostering the onset of neurodegenerative pathologies and inhibiting CNS regeneration, is also involved in neuropathic pain associated to traumas to the CNS. A large number of scientific publications report evident proofs of the fact that gliosis at spinal cord level is to be accounted for the onset and persistence of pathological pain related to inflammations, neuropathies and spinal immune activation. Indeed, it has been demonstrated in experimental models that reactive gliosis is necessary to create pathological pain states in laboratory animals.

Onset of gliosis following a CNS trauma seems to have, in the acute phase subsequent to the trauma, a reactive - protective effect, but later on it acts negatively by limiting CNS regeneration and function.

Moreover, it has also been reported that in the senescent brain a slight and progressive reactive gliosis

is present, which seems to be one of the factors inhibiting neurogenesis in the aging brain.

In spite of the evident pathological relevance of gliosis, therapeutic treatments that may induce the reversal of this phenomenon are not yet known. Substances able to inhibit glia activation do exist, but are not useful at a pharmacological level.

As it is also reported in the work by Watkins and Maier, (Nature reviews, 2, 2003, 973-85) , glia costitutes a new pharmacological target, and therefore it would seem desirable, given the multiple effect of gliosis, to identify substances and/or drugs able to oppose such a process in a preventive and/or curative manner, thereby preventing the onset of pathologies correlated thereto. Studies are reported in the literature with extremely discordant results on the use of NGF to counteract post-traumatic nerve pain. In many works, even very recent ones, the role of NGF remains controversial

(Sah et al, Nature reviews 2003, 2. 460-72) as its administration even seems to induce the appearance of neuropathic pain (allodynia and hyperalgesia) . In fact, it has been reported that NGF administration induces thermal hyperalgesia and that administration of an NGF receptor antagonist, ALE-0540, dose-dependently reduces mechanical allodynia. Other studies report that chronic intrathecal anti-NGF infusion has minimal effects on allodynia in neuropathic pain models. According to other studies, NGF restores the analgesic effect of opioids in chronic constriction injury. Anyhow, in spite of the extremely discordant results concerning NGF role in the induction of neuropathic pain, in the literature no cases are reported demonstrating the modulatory effect of NGF on reactive gliosis, described as central phenomenon also in chronic pain processes. SUMMARY OF THE INVENTION

In the present invention it was discovered that NGF or substances mimicking its function, when administered

in the site of the lesion, are able not only to prevent the appearance of reactive gliosis, but even to revert such a process, by restoring a no more hypertrophic glia, preventing or even making gliosis-correlated pathologies disappear in the involved compartment.

Therefore, the first object of the invention is the

NGF factor or its functionally active NGF-equivalent or

NGF agonist fragments or derivatives, for use in the curative and/or preventive therapeutic treatment of reactive gliosis.

The second object of the invention is the use of NGF, or its functionally active fragments or derivatives, i.e. NGF complete or partial agonists, or substances mimicking its function (agonists) , for the preparation of medicaments for the prevention and/or the treatment of reactive gliosis.

Object of the invention is also a therapeutic method comprising the administration, at pharmaceutically effective doses, of NGF or its functionally active derivatives or fragments, i.e. NGF agonists or partial agonists, or e# substances mimicking its function (agonists) , in or nearby the lesion.

DETAILED DESCRIPTION OF THE FIGURES

FIGURE Ia represents three histopathologic specimens of spinal cord removed from animals made neuropathic by sciatic nerve constriction and treated, in the left-to- right order, as disclosed in example 2, with NGF, ACSF, and, lastly, healthy control animals, on which analysis of the expression of the reactive gliosis marker, GFAP, was carried out.

The histogram reported below represents the respective quantitation of marker GFAP determination; values for the NGF-treated animal are similar to values measured on the healthy control animals, indicating that NGF inhibits the appearance of reactive gliosis in the animals, carrying the sciatic nerve constriction only.

FIGURE Ib represents three histopathologic specimens

of spinal cord removed from animals made neuropathic by sciatic nerve constriction, treated, in the left-to-right order, as described in example 2, with NGF, ACSF, and, lastly, healthy control animals on which it was carried out an analysis of the expression of the marker representative of the number of astrocytes, SlOOβ.

The histogram reported below represents the respective quantitation of the marker determination; values in the three cases are comparable. DETAILED DESCRIPTION OF THE INVENTION

According to the invention, NGF or its fragments or derivatives pharmacologically active as NGF agonists can be used for the regression and/or prevention of reactive gliosis and for the preparation of medicaments suitable therefor. Since reactive gliosis is a phenomenon preceding the onset of disease, NGF can be used for the prevention of the onset of pathologies that follow gliosis. Such pathologies comprise: stroke, injuries from trauma, growth of a tumor or neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, triplet diseases, amyotrophic lateral sclerosis, multiple sclerosis and neuropathic pain. In cases in which gliosis is the process necessary and sufficient for the development of symptoms associated to other pathologies, NGF can be used also for the cure of said manifestations. It can be used for human or animal use. For human use, murine NGF or recombinant human NGF (rhNGF)can be used, or functionally equivalent NGF fragments or derivatives or substances mimicking its function can be used. By "functionally equivalent derivatives" it is meant molecules capable of promoting at least one of the biological responses commonly associated with NGF. For example, compounds capable of supporting at least one of the biochemical or morphological NGF responses, such as interaction with its own receptors, and/or activation of TrkA receptor (auto- phosphorylation) , neuronal survival, or neurite

outgrowth, are defined as NGF agonists. By "functionally equivalent" [active] it is meant NGF fragments or derivatives maintaining the physiological function of NGF and that therefore might replace the native molecule. As recombinant NGF there may be used, for the purposes of the carrying out of the invention, the beta rhNGF disclosed in Pat. Appln. PCT/IB2005/053159 having the following characteristics.

The beta rhNGF disclosed in said patent application, and suitable for the carrying out of the invention, exhibits biological activities comparable to those exhibited by equal doses of 2.5S mNGF at least in the following tests: a. evaluation of PC12 pheochromocytoma cells differentiation into sympathetic-like neurons induced by incubation with the beta rhNGF of the invention, as compared to incubation with equal amounts of 2.5S mNGF; b. evaluation of survival and differentiation of dorsal root ganglia (DRG) explanted from 7-9 days old chick embryos and/or sympathetic paravertebral ganglia explanted from 10-12 days old chick embryos after incubation with the beta rhNGF 'of the invention, as compared to incubation with equal amounts of 2.5S mNGF; c. evaluation of phosphorylation of the high affinity TrkA receptor induced in PC12 cells by the beta rhNGF of the invention, as compared to equal amounts of 2.5S mNGF; and d. evaluation of the induction of hypertrophy of superior cervical ganglia, degranulation of mast cells and regulation of Substance P levels and high affinity TrkA receptor expression levels in cutaneous tissues of newborn mice treated with the beta rhNGF of the invention, as compared to equal amounts of 2.5S mNGF.

Said biological activities shall be higher than 76% of those given by equal doses of 2.5S mNGF in all the aforementioned tests. In particular, in test d. representing the in vivo activity of the molecule of the

invention, said activity will be advantageously comprised between 80 and 100% of the 2.5S mNGH used as reference.

In one particularly advantageous embodiment, said activity will be comprised between 90 and 100% in all the tests described above.

For the purposes of the invention, said tests can be carried out according to all the modes known to a person skilled in the art, provided that they are always performed by using equal amounts and/or concentrations (for example expressed as Molarity) of the beta rhNGF of the invention and 2.5S mNGF.

The beta rhNGF having the aforementioned properties and suitable for the carrying out of the present invention can be prepared according to a process comprising the following steps: i) the construction of an expression vector suitable for expression in mammalian cells and comprising a cDNA sequence encoding the exon 3 of the human NGF gene, said cDNA sequence including a sequence encoding the beta chain of mature human NGF (120 aa) , a sequence encoding the prosequence of the beta chain of human NGF (103 aa) and a sequence encoding the signal sequence of the beta chain of human NGF (18 aa) ; ii) the transformation of mammalian cells with said vector; iii) the selection of cellular clones able to secrete beta rhNGF having biological activities higher than 76% of those given by the 2.5S subunit of the native murine NGF in the following tests: a. evaluation of PC12 pheochromocytoma cells differentiation into sympathetic-like neurons induced by incubation with the beta rhNGF of the invention, as compared to incubation with equal amounts of 2.5S mNGF; b. evaluation of survival and differentiation of dorsal root ganglia (DRG) explanted from 7-9 days old chick embryos and/or sympathetic paravertebral ganglia explanted from 10-12 days old chick embryos after

incubation with the beta rhNGF of the invention, as compared to incubation with equal amounts of 2.5S mNGF; c. evaluation of phosphorylation of the high affinity TrkA receptor induced in PC12 cells by the beta rhNGF of the invention, as compared to equal amounts of 2.5S mNGF; d. evaluation of the induction of hypertrophy of superior cervical ganglia, degranulation of mast cells, and regulation of Substance P levels and high affinity TrKA receptor expression levels in cutaneous tissues of newborn mice treated with the beta rhNGF of the invention, as compared to equal amounts of 2.5S mNGF; iv) cultivation of the cells selected at point iii) and recovery of said beta rhNGF directly from the culture medium.

Here are provided some of the abbreviations used for this description: rhNGF= recombinant human Nerve Growth Factor; mNGF= murine Nerve Growth Factor (murine 2.5S NGF or beta-NGF) ;

TrkA= high affinity NGF receptor; DRG= Dorsal Root Ganglia; SCG= Superior Cervical Ganglion; SP= Substance P. Sympathetic-like neurons = cells exhibiting some characteristics of sympathetic neurons, such as neurite processes similar to those of the sympathetic neurons and some neurotransmitters like dopamine and norepinephrine.

The biological activity in vitro can be analyzed by using PC12 pheochromocytoma cells (Greene LA & Tischler

AS, Proc. Natl. Acad. Sci. USA 73: 2424-2428, 1976). Said cells represent the in vitro neuronal system generally used to analyze the NGF signal transduction and the biochemical and morphological responses induced by this neurotrophic factor.

Another neuronal system is represented by both dorsal root ganglia (DRG) prepared from 7, 8 or 9 days

old chick embryos and paravertebral sympathetic ganglia prepared from 10, 11 or 12 days old chick embryos.

The test at point a. may be carried out by- evaluating the differentiation of the aforementioned PC12 cells into sympathetic-like neurons with formation of long neurite processes. Differentiation can be expressed, for example, as percentage of the number of cells with neurite processes within a certain interval of time, or in terms of effectiveness on differentiation always within a certain time, as compared to a control system. In the present invention, the activity of the 2.5S mNGF in the same experimental conditions was used as positive control, i.e. maximum NGF activity equal to 100%.

The differentiation response can be induced after incubation of cells with concentrations of beta rhNGF, or 2.5S mNGF, comprised between 1 and 100 ng/ml, in particular, between 5 and 20 ng/ml, and can be observed after an incubation time between 8 and 48 hours, in particular for a time comprised between 16 and 24 hours. In a further embodiment of the invention, the test at point b. could be carried out on explants of dorsal root ganglia (DRG) prepared from 7-9 days old chick embryos and/or explants of paravertebral sympathetic ganglia of 10-12 days old chick embryos, by evaluating ' the survival and differentiation of said explants in the presence of concentrations of rhNGF, or 2.5S mNGF, comprised between 1 and 100 ng/ml and after an incubation time comprised between 24-48 h. Differentiation and survival can be maintained in the presence of said concentrations of rhNGF up to about 2-3 weeks.

The test at point c. could be carried out by evaluating the phosphorylation of the high affinity TrkA receptor by immunoprecipitation experiments. The peak of receptor activation can be observed after short times between 1 and 10 min, but in particular after 5 min, and with concentrations of rhNGF, or 2.5S mNGF, comprised between 1 and 100 ng/ml, for example concentrations of 5-

10 ng/ml could be used.

The biological activity of the rhNGF at issue may be analyzed both in the conditioned culture medium of the rhNGF-producing cells properly diluted in the culture medium of PC12 cells or in ganglia to give the desired final concentrations. It can also be analyzed in the medium of the middle/large scale production systems in order to verify that the biological activity of the rhNGF produced in said systems is comparable, according to the indications given above, to those of the 2.5S mNGF. Production systems suitable for middle or large scale can be, for example, commercially available production systems like MiniPERM® (Greiner Bio-One, Germany) , Roller Bottles or any other bioreactor system known to the technician of the field for middle or large scale production of recombinant proteins in mammalian cells growing in adhesion.

For the assay at point d. regarding the in vivo biological activity, newborn mice can be used. After injection of equal doses of beta rhNGF or 2.5S mNGF, usually about 5 μg/gr of body weight for 5 consecutive days in parallel experiments, the biological activity of the recombinant neurotrophin produced on: superior cervical ganglia (hypertrophy) and cutaneous tissue at the injection site (mast cell activation and regulation of Substance P and TrkA levels) can then be evaluated.

Concerning the hypertrophy of SCG ganglia, a histological analysis can be carried out, comparing the effect of the beta rhNGF used to that of the murine neurotrophin by standard techniques for preparation fixing and staining, well known to the technician of the field, such as for example toluidine blue staining.

To produce the rhNGF disclosed in the aforementioned patent application, the cDNA of interest may be cloned by means of standard PCR techniques using, for example, primers that can be obtained with standard programs, able to amplify the region of interest (exon 3) , using the

published human NGF sequences to design the primers. Then, the cDNA of interest (exon 3 of the human NGF gene, published in the literature, e.g., Ullrich et al . , Nature 303: 821-825, 1983) can be inserted into vectors that allow the verification of the correct sequence of the insert which shall comprise the sequence coding for the 120 aa of the mature human NGF, the sequence coding for the 103 aa of the prosequence present in the human proNGF, and the sequence coding for the 18 aa of the signal sequence of the native human NGF. Said cDNA could be subsequently sub-cloned into an appropriate vector.

The vector may be any vector known in the literature and/or commercially available able to express the inserted protein in mammalian cells. Among these: the pTRE vector (TetOff System, Clontech) or any other vector comprising a strong inducible promoter, such as for example the vectors of the pT-REx series (Invitrogen) . The choice of a vector containing a strong promoter, such as for example the CMV promoter, offers the advantage of guaranteeing high production of the protein of interest in eukaryotic cells. In particular, a tetracycline- regulated vector, guarantees maximum expression levels, much higher than those that can be obtained with a vector containing a constitutive CMV promoter. For instance, the pTRE vector (TetOff System, Clontech) contains, upstream of the minimal CMV promoter, seven repeats of a tetO sequence for binding of the regulatory protein tTA encoded by the regulatory pTet-Off plasmid (Gossen M & Bujard H, Proc. Natl. Acad. Sci . USA 89: 5547-5551, 1992) . This regulatory system ensures expression levels of the recombinant protein even higher than o'ther inducible expression systems containing, beside the promoter, enhancer regions that are responsive to heavy metals or steroid hormones . Other possible vectors suitable for expression in mammalian cells include the RheoSwitch system (NewEngland BioLabs), macrolide-inducible vectors, such as pTRIDENT,

pDuoRex, pMFl89, pMF229 (Weber W et al., Biotechnol. Bioeng. 80: 691, 2002), ecdysone-inducible vectors such as the pEGSH (Stratagene) .

In one embodiment of the invention, the amplified construct described above can be subcloned in a pTRE vector, (TetOff System, Clontech) , downstream of the pCMV promoter present in the commercial vector, thus generating the pTRE-hNGF construct .

The mammalian cells for the cloning can belong to any mammalian cell line, known to the skilled person, suitable for production of human proteins. Among these, merely by way of a non-limiting example, are the HeLa, MEF, CHO, COS, BHK, HEK293, VERO cells, W138 and MDCK cell lines, or L929 fibroblasts, 3T3 fibroblasts, or other stabilized mammalian cell lines. Anyhow, whichever is the cellular system used, the cells shall be genetically modified to constitutively express, besides the plasmid vector comprising the human NGF cDNA, also the regulatory protein required by the inducible system of choice. Transformation of the mammalian cells with the appropriate expression vector as indicated above can be carried out by any one of the transformation methods known to the technician of the field, such as for example, electroporation, transfection by calcium- phosphate precipitation or liposomal complexes.

The selection of suitable cells could be performed by verifying the abundant presence of beta rhNGF in the culture medium, and by analyzing said beta rhNGF with the tests indicated above. Thus, the clones obtained coul be selected depending on the properties of the beta rhNGF produced and their ability to secrete said beta rhNGF.

The beta rhNGF described in the aforementioned patent could be recovered, according to the production process of the invention, directly from the cell culture medium without the need of extraction from cells and thus greatly limiting the likelihood of contamination of the protein with cellular materials, such as, for example,

unprocessed forms of NGF. The protein so obtained can be, when required, purified by means of standard techniques known to the expert of the field.

As NGF derivative or fragment, there can be used, e.g., the peptides disclosed in European Patent Appln. No. 05012643.2.

Said peptides, having a structure characterized by the presence of two loops constrained in cyclic structure by the presence of covalent bonds between amino acid side chains, wherein the amino acid sequences of the first and second loop of said molecules are, respectively, substantially homologue to those of loop 1 (loop 1, residues 29-38) and of loop 4 (loop 4, residues 92 and 97) of NGF. Said peptides can be represented by formula

I H ₁ I I

(Xaa)n- AAl- (Yaa)m-AA2-L-AA3- (Zaa)m-AA.4

Wherein: AAl and AA2 are selected from the group comprising Cys, Asp, GIu, Lys, Orn, Pen or Dap and are linked through an amide or S-S bond between chemical functions on their side chains;

AA3 and AA4 are selected from the group comprising Cys, Asp, GIu, Lys, Orn, Pen or Dap and are linked through an amide or S-S bond between chemical functions on their side chains;

L is a linker sequence formed by 2-4 amino acid residues, an organic linker or a mixed sequence formed by amino acid residues and organic linker;

(Yaa)m is an amino acid sequence formed by 4 - 8 residues;

(Zaa)m is an amino acid sequence formed by 4 - 8 residues; (Xaa)n is an amino acid sequence in which n is an integer between 0 and 22.

For the purposes of the carrying out of the present

invention, L may be the TGA sequence. An example of a suitable organic linker is the di-oxoethylene group.

(Yaa)m may be a sequence homologue to that of loop 1 (residues 29-38) of NGF, like, e.g., the sequence TDIKGK. (Zaa)m may be a sequence homologue to that of loop 4 (residues 92-97) of NGF, like, e.g., the sequence DGKQ.

When n is not zero, (Xaa)n may represent a sequence homologue to that of N-terminus of NGF, e.g. the sequence HPIFHRGEFSVADSVSVWVGD . The aforeindicated peptides may be free or acetylated in the N-terminal position, they may be in carboxylic free form or in amide form in the C-terminal position; one, two or more amino acid residues may be added on the C-terminal end. The position of the first and the second loop along the polypeptide chain can also be reversed. Said peptides may also be a dimeric form of those described in the above formula. In this case, a covalent bond is present between the monomeric peptides . For the carrying out of the present patent application, NGF or a fragment or derivative thereof having NGF agonist function is formulated in a composition suitable for administration in the compartment or in the neighbourhood of the glial compartment of interest.

It may be administered topically, subcutaneously, intravenously, intraventricularly, intrathecally, intranasally, by aerosol or ophthalmic eyewash for CNS- involving glioses, as it has been demonstrated ^• that NGF administered by aerosol is able to pass through the hematoencephalic barrier, by liposomes, or even by gene therapy by the insertion of suitable molecules transformed so as to express said molecule. NGF may also be administered conjugated to antibodies such as an antibody directed against the transferrin receptor (OX- 26) abundantly expressed on the hemato-encephalic barrier and capable of mediating its transit.

For administration: topically, subcutaneously, intravenously, intraventricularly, intrathecally, intranasally, by areosol or by ophthalmic eyewash, the NGF or its derivatives indicated above could be prepared by standard formulations ^' known to the technician of the field at pharmacologically effective dosages with suitable excipients.

The methods of preparing formulations as those indicated above are known and available in the literature, and it is not necessary to provide further indications for their realization. For each of these administration ways there are also reported in the literature examples precisely referring to NGF-comprising preparations. Also for intrathecal administration systems are known, such as the baclofen infusion pump, suitable for such an administration.

Concerning gene therapy, there may be used cells such as fibroblasts, stem cells and other suitable cells genetically modified in order to express NGF. For example, there may be used an expression system adjustable in vivo by using a tetracycline-regulated promoter ("TetOff system") as the aforedescribed one.

For administration by liposomes, there may be realized NGF-containing liposomes as those described by Xie et al . 2005, (J. Control release 105.1-2, pplO6-119) . Administration should be performed so as to provide a pharmacologically effective dose of the molecule.

Concerning dosage, a weight-proportional dose can be hypothesized, owing to the diversity of drug volume of distribution between animal and human. Moreover, dosage also varies depending on the administration means used, since pharmacokinetics (drug metabolization) varies.

The ^' following examples aim at illustrating the invention in a non-limiting manner and demonstrating NGF effect on reactive gliosis.

EXAMPLES : Example 1.

Evidence of presence of reactive gliosis resulting in peripheral neuropathy in rats.

Experiments were performed on male Sprague Dawley rats (250-300 g) . Rats were treated so as to induce in them a sciatic nerve constriction with consequent peripheral neuropathy.

Presence of reactive gliosis was measured by evaluating in situ GFAP expression. The deeply anesthesized animals were perfused cardially with saline solution (0.1M TRIS HCl, 10 mM EDTA) followed by 4% paraformaldehyde added to 0.1% glutaraldehyde in 0.01 M phosphate-buffer (PB), pH 7.4 at 4°C. Removed spinal cords were post-fixed two hours in the same fixative, balanced in 30% sucrose PBS and cryoconserved in chilled isopentane on dry ice. 25-μm thick serial sections obtained at the cryostat were first treated with 10% ^' normal serum in PB added with 0.25% Triton® for Ih. Then, the sections were reacted with an anti-GFAP antibody

(1:1000, Sigma, Milan, Italy), followed by a specific biotinylated secondary antibody, highlighted by Vectastain® system (Vector Labs, Inc., Burlingame, CA, USA), and reacted with 3, 3_-diaminobenzidine tetrahydrochloride (DAB; Sigma, 0.5 mg/ml Tris-HCl) and 0.01% hydrogen peroxide. The sections, mounted on slides, were studied on a Zeiss Axioskόpe 2 light microscope connected to a high-resolution digital camera (C4742-95, Hamamatsu Photonics, Italy) . Densitometric analysis of GFAP expression in the dorsal horn of the spinal cord was accomplished by using a computer-assisted image analysis system (MCID 7.0; Imaging Res. Inc, Canada). The value expressed the total target area as relative percent to the scanned area. The averages were obtained from five randomly selected spinal cord sections for each animal, and compared with the values of control groups. The data were exported and converted to a frequency distribution histogram using the Sigma-Plot 8.0 program (SPSS Erkrath Germany) . The NGF group was compared to the control group

by a paired t-test .

Mechanical sensitivity of the animals (allodynia test) was evaluated by measuring the time of hind paw withdrawal response to von Frey filament stimulation. Thermal sensitivity of the animals (hyperalgesia test) was evaluated by measuring the time of withdrawal latency from noxious heat source according to the Plantar test method.

Example 2. Reactive gliosis presence, or lack thereof, in peripherally neuropathic rats treated with NGF or ACSF only.

Seven-day peripherally neuropathic rats were implanted with osmotic system Alzet 2001® (Alza Corp., Cupertino, CA, USA) . The system held a solution of artificial cerebral spinal fluid (ACSF) , as vehicle containing 1 mg/ml rat serum albumin and rat recombinant beta-NGF at a concentration of 125 ng/μl. The osmotic pump released the content at a rate of 1 μl/h for 7 days. Thus, an intrathecal infusion equal to 125 μg/h was produced. The system was attached to a PElO catheter, the catheter was inserted into the subarachnoid space, to the lumbar enlargement.

There were treated also rats in parallel with the same system, but with administration of ACSF only, called ACSF group rats.

Mechanical sensitivity of the animals (allodynia test) was evaluated by measuring the time of hind paw withdrawal response to von Frey filament stimulation. Thermal sensitivity of the animals (hyperalgesia test) was evaluated by measuring the time of withdrawal latency from noxious heat source according to the Plantar test method.

Seven days after induction of the injury and following seven days of the NGF (or ACSF only) treatment, gliosis reached its maximum expression in control animals only (ACSF group) . Fourteen days after induction of the

injury and following seven days of the NGF (or ACSF only) treatment (yielded data are reported in FIGURE Ia) , rats belonging to the ACSF group showed, in the dorsal horn of the spinal cord, a marked gliosis as expressed by an intense reaction for GFAP (2.345 ± 0.16) when compared to the modest reaction found in NGF group rats (1.365 + 0.16) therefore not showing any difference with a CTR group of control rats (1.44 ± 0,15) .

The marked gliosis found in the dorsal horn of the spinal cord of the ACSF group is due to a transformation of the astrocytes from the protoplasmic to the fibrillary type, a reaction due more to the hypertrophy of single cells rather than to a significant increase of the total number of glial cells, as showed by the analysis of SlOOβ expression (yielded data are reported in FIGURE Ib) . In fact, in the ACSF group, the SlOOβ expression is only slightly higher compared to that of the group treated with NGF and the CTR group (1.945 ± 0.15 and 1.82 ± 0.23 and 1.56 ± 0.2, respectively).