Liposome-mediated transfection of central nervous system cells

Disclosed are methods for use in transferring nucleic acids into central nervous system cells in vivo and in vitro and/or for stimulating central nervous system cells. Neurotrophic genes are shown to stimulate neurofilament cells and to promote nerve cell growth, repair and regeneration in vivo. Gene transfer protocols are disclosed for use in transferring various nucleic acid materials into central nervous system cells, as may be used in treating various pathologies of the brain and spinal cord.

BACKGROUND OF THE INVENTION 
A. Field of the Invention 
The present invention relates generally to the field of molecular biology. 
More particularly, certain embodiments concern the transfer of genetic 
material into central nervous system cells. In certain examples, the 
invention concerns the use of liposome-mediated delivery of nucleic acids 
encoding neurotrophic factors to stimulate nervous system cell repair and 
regeneration. Rescue of neurofilament protein following traumatic brain 
injury (TBI) in vivo has been demonstrated. 
B. Description of the Related Art 
1. Neurotrophins Play Important Role in Cortical Injury Recovery 
Brief depolarization of primary septo-hippocampal cell cultures can produce 
significant losses of neurofilament proteins. Studies have indicated that 
brain-derived neurotrophic factor (BDNF) increases neurofilaments in 
hippocampal cell cultures (Yip et al., 1993) and increases survival of 
cortical neurons (Ghosh et al., 1994). 
Several articles have reported attempted treatments for treating traumatic 
brain injury. Exogenous supplementation of NGF has been reported to spare 
neurons from death and degeneration following injury (Hafti, 1986; Kromer, 
1987; Montero and Hafti, 1988; Williams et al., 1986) and to increase 
choline acetyl transferase (CHAT) activity (Rylett et al., 1993; Williams 
and Rylett, 1990). 
NGF prevents degeneration of septal cholinergic neurons following fimbria 
lesions or transfection (see e.g., Morse et al., 1993). NGF and BDNF 
increase survival of septal cholinergic neurons in vitro (see e.g., 
Alderson et al., 1990) and increase choline acetyl transferase (ChAT) 
activity both in vitro (see e.g., Alderson et al., 1990) and in intact 
animals (see e.g., Rylett et al., 1993). 
Observations indicate that TBI can result in disturbances of cholinergic 
neurotransmission associated with impaired release of acetylcholine in the 
hippocampus (Dixon et al., 1993). In some cases, this disruption may cause 
nerve death, which may ultimately lead to brain dysfunction. However, the 
mechanism of cholinergic disturbance is not well-understood, and therapies 
addressing this abnormality in vivo have not been reported. 
2. NGF and BDNF Expression in the Hippocampus 
NGF and BDNF are members of the neurotrophin gene family, as are 
neurotrophin-3, and neurotrophins-4/5 (NT-3, NT-4/5). All are expressed in 
brain (for review, see Lindsay, 1993). During development in the adult rat 
brain BDNF and NGF mRNA are particularly abundant in the hippocampus 
(Maisonpierre et al., 1990b; Ernfors et al., 1990), a region which is 
preferentially vulnerable to TBI (Hayes et al., 1992; Olenik et al., 
1988). 
3. BDNF and NGF Blunt Free Radical Damage 
Formation of free radicals and subsequent lipid peroxidation may contribute 
to TBI (Chan, 1992; Hall et al., 1992). Giving NGF to adult rats protects 
their sympathetic ganglia from 6-hydroxydopamine toxicity, a free radical 
generator (Johnnson, 1980). Furthermore, in adult rats, long-term NGF 
administration increases the activity of antioxidant enzymes in cortex 
(Nistico et al., 1992). Protection by NGF in cultures of PC-12 cells is 
associated with specific increases in catalase activity and glutathione 
levels due to stimulation of glutathione redox reactions and synthesis 
(see e.g., Sampath et al., 1993). 
4. BDNF and NGF Initiate Cytoskeleton Repair 
NGF has been shown to initiate and maintain neurite outgrowth in rat 
pheochromocytoma (PC12) cells (Greene and Tischler, 1982), and is 
associated with increased levels of .beta.-tubulin (see e.g., Teng and 
Greene, 1993). BDNF has been shown to increase in vitro levels of 
neurofilament in hippocampal cell cultures (Yip et al., 1993). However, 
the role of these two growth factors in cytoskeletal stabilization in vivo 
had not been determined. 
5. BDNF and NGF Are Involved in Neurodegenerative Disease 
TBI is a risk factor for Alzheimer's disease (Mortimer et al., 1991), and 
in some cases, is also associated with diffuse deposition of 
.beta./A.sub.4 protein (Roberts et al., 1991; Clinton et al., 1991), the 
amyloidogenic protein of Alzheimer's disease. Furthermore, in Alzheimer's 
disease in situ hybridization reveals significant decreases in BDNF mRNA 
(Phillips et al., 1991). In aged rats, NGF increases high affinity choline 
transport (Williams et al., 1990), stimulates Ach release (Rylett et al., 
1993) and also improves the performance of age-impaired rats in spatial 
memory tasks (Fischer et al., 1987). 
6. BDNF and NGF Expression After CNS Injury is Only Transient 
Although various injuries to the CNS up-regulate the production of 
neurotrophins and increase BDNF and NGF mRNA in the hippocampus (Yang et 
al., 1993), in most injuries, production of neurotrophins is not sustained 
long enough to promote recovery. In TBI, prominent up-regulation of BDNF 
mRNA occurs in the hippocampus, but only transiently, from 1 h to 6 h 
after injury (Yang et al., 1993), and therefore levels of neurotrophins 
are not present in substantial levels to effect cytoskeletal (e.g., 
neurofilament) rescue. 
7. Providing Neurotrophins to CNS Cells is Desirable Following TBI 
Many different approaches have been contemplated to deliver exogenous 
neurotrophins to the nervous system of mammals (Hafti, 1986; Kromer, 1987; 
Montero and Hafti, 1988; Williams et al., 1986), although significant 
limitations imposed by protein degradation and by the blood-brain barrier 
have restricted the clinical utility of these approaches (Barinega, 1994). 
Unfortunately, even though exogenous growth factors can be delivered within 
the brain by means of mini-osmotic pumps that release small amounts of 
neurotrophin into the ventricular cavity or directly into the parenchyma 
(Hafti, 1986; Williams et al., 1986; Kromer, 1987; Montero and Hafti, 
1988), pumps must be mechanically regulated, and often can fail. This 
method is particularly expensive and inconvenient, since the stored growth 
factors in the pump reservoir diminish in activity over time, and fresh 
amounts of the neurotrophin must be constantly added. 
Many drawbacks are also associated with these types of treatment protocols, 
not the least of which is the expensive and time-consuming purification of 
the recombinant proteins from their host cells (Rylett et al., 1993; Morse 
et al., 1993). Also, polypeptides, once administered to an animal are more 
unstable than is generally desired for a therapeutic agent, and they are 
susceptible to proteolytic attack. Furthermore, the administration of 
recombinant proteins can initiate various inhibitive or otherwise harmful 
immune responses. 
8. Focal Delivery of Neurotrophins 
Investigators studying central nervous system injury have long recognized 
that changes in specific proteins may be important determinants of 
pathological responses to injury and recovery of function of the injured 
brain. Gene transfer has emerged as a potential way to introduce 
neurotrophins into CNS cells and tissues (Friedmann and Ginnah, 1993). 
Methods currently used to introduce genes into localized regions of the 
nervous system through stereotactic injection include retroviral vectors, 
herpes virus vectors, adenoviral vectors and grafted cells. Grafted cells 
have been most extensively examined for growth factor production. 
Studies grafting cells in brain capable of producing growth factors have 
employed mouse sarcoma cells, male mouse submaxillary gland cells 
(Levi-Montalcini and Cohen, 1960; Caramia et al., 1962) and sciatic nerve 
cells (Richardson and Ebendal, 1982) or alternatively, in cell culture 
followed by implantation within the brain (Gage et al., 1987). Despite 
limited success with several different cell types, including primary 
fibroblasts (Kawaja et al., 1992), astrocytes and immortalized cell lines 
(Rosenberg et al., 1988; Wolf et al., 1988) grafts remain only a temporary 
solution, since most will eventually be rejected. 
Although retroviral vectors are considered the most efficient vectors for 
stable gene transfer into mitotic mammalian cells and have been widely 
used in CNS cancer gene therapy (Yamada et al., 1992; Ram et al., 1993; 
Culver et al., 1992), they are unsuitable for post-mitotic CNS cells, 
since with the exception of some lentiviruses, retroviruses can integrate 
only into chromosomes of dividing cells. Another significant limitation of 
retroviral vectors is their maximum insert capacity of 5-7 kb (Gelinas and 
Teman, 1986). 
Two methods using viral vectors have been described: 1) Herpes Simplex 
virus, and 2) adenovirus-mediated delivery systems. Unfortunately, 
however, neither are suitable for treatment of TBI. Although Herpes virus 
is able to infect post-mitotic cells and can be taken up anywhere along 
the cell surface (Breakefield and DeLuca, 1991; Lycke et al., 1988), its 
toxicity to nervous cells (Johnson et al., 1992) and its disruption of 
normal neuronal architecture (Huang et al., 1992) make it unsuitable for 
treatment of TBI and CNS cells. 
Replication-deficient adenoviral vectors have also been used to infect rat 
CNS cells in vitro and in vivo, since these DNA viruses are able to infect 
post-mitotic cells (le Gal La Salle et al., 1993), but unfortunately, they 
do not integrate efficiently into the nuclear DNA of the recipient cells, 
gene expression occurs only transiently, often in an unpredictable manner 
(Graham and Prevec, 1991; Horwitz, 1990). Further limiting these methods 
in the treatment of TBI is their limited transfection into neural cells, 
and the pathogenicity and limited duration of gene expression of these 
vectors. 
Several groups have investigated the possibility of using liposomes as a 
means of mediating delivery of genetic information into nervous system 
cells in vitro, but unfortunately these methods were disappointing. For 
example, using the E. coli .beta.-Gal reporter gene in liposome-mediated 
transfection of low density cultures of rat hippocampal neurons, the 
transfection efficiency was less than 1% of the whole cell population, and 
the small fraction of transfected cells was mostly neuronal (Drazba and 
Ralston, 1993). 
Likewise, an ex vivo method employing cationic liposomes to transfect 
primary rodent neuronal cell cultures with a gene encoding .beta.-Gal (Ono 
et al., 1990) showed only limited success, when the cell types which 
incorporated and expressed the injected cDNA were not delineated. 
9. Deficiencies in the Prior Art 
A method of treating a variety of central nervous system pathologies 
through manipulations of specific trophic and/or toxic proteins would have 
important therapeutic potential. Increasing expression of trophic factors 
which have been shown to enhance recovery of function following trauma to 
the brain and spinal cord would be particularly desirable. More than 
500,000 patients annually are hospitalized for traumatic brain injury. 
More than 10,000 patients are treated for spinal cord injury and 750,000 
patients for stroke. Neurodegenerative diseases, organically-based 
psychological disorders and chronic pain are all widely recognized major 
health problems in the United States. The presence of the blood-brain 
barrier significantly confounds the choice of routes for administration of 
neurotrophins. 
It is clear, therefore, that a new method capable of promoting nervous 
system cell repair and regeneration in vivo would represent a significant 
scientific and medical advance with immediate benefits to a large number 
of patients. A method readily adaptable for use with a variety of growth 
factors and other genes would be particularly advantageous. Rescue of 
neurofilament loss due to TBI by increasing the availability of 
neurotrophins both ex vivo and in vivo would represent a significant 
improvement in the treatment of central nervous system injury. 
SUMMARY OF THE INVENTION 
The present invention overcomes one or more of these and other drawbacks 
inherent in the prior art by providing novel methods for use in 
transferring nucleic acids into post-mitotic nervous system cells and 
tissues, and for promoting nervous system cell repair and regeneration. 
Certain embodiments of the invention rest, generally, with the inventors' 
surprising finding that nucleic acids can be effectively transferred to 
post-mitotic cells by liposome-mediated transfection protocols both in 
vivo and ex vivo. Moreover, in certain embodiments, the transfer of a 
neurotrophic gene stimulates nervous system cell repair in an animal. 
The invention, in general terms, thus concerns methods for transferring a 
nucleic acid segment into neural cells or tissues. The methods of the 
invention generally comprise contacting neural cells or tissues with a 
composition comprising a nucleic acid segment in a manner effective to 
transfer the nucleic acid segment into the cells. 
Alternatively, the neural cells may be located within a central nervous 
system (CNS) tissue site of an animal, when the nucleic acid composition 
would be applied to the site in order to effect, or promote, nucleic acid 
transfer into CNS cells in vivo. In transferring nucleic acids into CNS 
cells within an animal, a preferred method involves first adding the 
genetic material to a liposome complex and then using the liposome-DNA 
complex to transfect an appropriate tissue site within the animal. 
An extremely wide variety of genetic material can be transferred to CNS 
cells or tissues using the compositions and methods of the invention. For 
example, the nucleic acid segment may be DNA (double or single-stranded) 
or RNA (e.g., mRNA, tRNA, rRNA); it may also be a "coding segment", i.e., 
one that encodes a protein or polypeptide, or it may be an antisense 
nucleic acid molecule, such as antisense RNA that may function to disrupt 
gene expression. The nucleic acid segments may thus be genomic sequences, 
including exons or introns alone or exons and introns, or coding cDNA 
regions, or in fact any construct that one desires to transfer to a CNS 
cell or tissue. 
Suitable nucleic acid segments may also be in virtually any form, such as 
naked DNA or RNA, including linear nucleic acid molecules and plasmids; 
functional inserts within the genomes of various recombinant viruses, 
including viruses with DNA genomes and retroviruses; and any form of 
nucleic acid segment, plasmid or virus associated with a gold particle 
which may be employed in connection with the gene gun technology. 
The invention may be employed to promote expression of a desired gene in 
CNS cells or tissues and to impart a particular desired phenotype to the 
cells. This expression could be increased expression of a gene that is 
normally expressed (i.e., "over-expression"), or it could be used to 
express a gene that is not normally associated with CNS cells in their 
natural environment. Alternatively, the invention may be used to suppress 
the expression of a gene that is naturally expressed in such cells and 
tissues, and again, to change or alter the phenotype. Gene suppression may 
be a way of expressing a gene that encodes a protein that exerts a 
down-regulatory function, or it may utilize antisense technology. 
1. Central Nervous System Cells and Tissues 
In certain embodiments, this invention provides advantageous methods for 
using genes to stimulate CNS cells. As used herein, the term "CNS cells" 
refers to any or all of those cells that have the capacity to ultimately 
form, or contribute to the formation of, central nervous system tissue. 
This includes various cells in different stages of differentiation, such 
as, for example, developmentally different fetal and adult neural cells, 
as well as neurons, astroglia, microglia, and oligodendrocytes, and the 
like. CNS cells also include cells that have been isolated and manipulated 
in vitro, e.g., subjected to stimulation with agents such as cytokines or 
growth factors or even genetically engineered cells. The particular type 
or types of CNS cells that are stimulated using the methods and 
compositions of the invention are not important, so long as the cells are 
stimulated in such a way that they are activated and, in the context of in 
vivo embodiments, ultimately give rise to CNS cells and tissue. CNS cells 
may also be isolated from animal or human tissues and maintained in an in 
vitro environment. Isolated cells may be stimulated using the methods and 
compositions disclosed herein and, if desired, be returned to an 
appropriate site in an animal where CNS cell repair is to be stimulated. 
In such cases, the nucleic-acid containing cells would themselves be a 
form of therapeutic agent. Such ex vivo protocols are well known to those 
of skill in the art. 
In important embodiments of the invention, the CNS cells and tissues will 
be those cells and tissues that may be damaged and that one desires to 
treat. Accordingly, in treatment embodiments, there is no difficulty 
associated with the identification of suitable target cells to which the 
present therapeutic compositions should be applied. All that is required 
in such cases is to obtain an appropriate stimulatory composition, as 
disclosed herein, and contact the site of the injury or defect with the 
composition. The nature of this biological environment is such that the 
appropriate cells will become activated in the absence of any further 
targeting or cellular identification by the practitioner. 
Certain methods of the invention involve, generally, contacting CNS cells 
with a composition comprising one or more neurotrophic genes (with or 
without additional genes, proteins or other biomolecules) so as to promote 
expression of said gene in said cells. As outlined above, the cells may be 
contacted in vitro or in vivo. This is achieved, in the most direct 
manner, by simply obtaining a functional neurotrophic gene construct and 
applying the construct to the cells. The inventors surprisingly found that 
there are no particular molecular biological modifications that need to be 
performed in order to promote effective expression of the gene in CNS 
cells. Contacting the cells with a liposome complex containing a suitable 
DNA molecule, e.g., a linear DNA molecule, or DNA in the form of a plasmid 
or other recombinant vector, that contains the gene of interest under the 
control of a promoter, along with the appropriate termination signals, is 
sufficient to result in uptake and expression of the DNA, with no further 
steps being necessary. 
2. Neurotrophic Genes 
As used herein, the term "neurotrophic gene" is used to refer to a gene or 
DNA coding region that encodes a protein, polypeptide or peptide that is 
capable of promoting, or assisting in the promotion of, nerve cell 
formation, or one that increases the rate of primary nerve cell growth or 
healing (or even a gene that increases the rate of neurofilament tissue 
growth or healing). The terms promoting, inducing and stimulating are used 
interchangeably throughout this text to refer to direct or indirect 
processes that ultimately result in the formation of new CNS tissue or in 
an increased rate of CNS cell repair. Thus, a neurotrophic gene is a gene 
that, when expressed, causes the phenotype of a cell to change so that the 
cell either differentiates, stimulates other cells to differentiate, 
attracts CNS-forming cells, or otherwise functions in a manner that 
ultimately gives rise to new CNS tissue. 
A variety of neurotrophic genes are now known, all of which are suitable 
for use in connection with the present invention. Neurotrophic genes and 
the proteins that they encode include, for example, BDNF, NGF, fibroblast 
growth factor (FGF), insulin-like growth factor, glial cell line-derived 
neurotrophic factor, ciliary neurotrophic factor, endothelial growth 
factor (EGF); chemotactic or adhesive peptides or polypeptides; 
morphogenetic proteins; and even growth factor receptor genes. Any of the 
above or other related genes, or DNA segments encoding the active portions 
of such proteins, may be used in the novel methods of the present 
invention. 
As known to those of skill in the art, the original source of a recombinant 
gene or DNA segment to be used in a therapeutic regimen need not be of the 
same species as the animal to be treated. In this regard, it is 
contemplated that any recombinant growth factor (GF) or neurotrophic gene 
may be employed to promote CNS cell repair or regeneration in a human 
subject or an animal, such as, e.g., a horse. Particularly preferred genes 
are those from human. However, since the sequence homology for genes 
encoding nerve growth factors is highly-conserved across species lines, 
rodent and bovine species may also be contemplated as sources, in that 
such genes and DNA segments are readily available, with the human or 
rodent forms of the gene being most preferred for use in human treatment 
regimens. Recombinant proteins and polypeptides encoded by isolated DNA 
segments and genes are often referred to with the prefix "r" for 
recombinant and "rh" for recombinant human. As such, DNA segments encoding 
rGFs, such as rhNGF or rhBDNF, etc. are contemplated to be particularly 
useful in connection with this invention. Any recombinant neurotrophic 
gene would likewise be very useful with the methods of the invention. 
The definition of a "GF gene", as used herein, is a gene that hybridizes, 
under relatively stringent hybridization conditions (see, e.g., Maniatis 
et al., 1982), to DNA sequences presently known to include GF gene 
sequences. 
The definition of a "neurotrophic gene", as used herein, is a gene that 
hybridizes, under relatively stringent hybridization conditions (see, 
e.g., Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual, Cold 
Spring Harbor Laboratory), to DNA sequences presently known to include 
neurotrophic gene sequences. 
To prepare a neurotrophic gene segment or cDNA one may follow the teachings 
disclosed herein and also the teachings of any of patents or scientific 
documents specifically referenced herein. One may obtain a hGF or 
neurotrophic gene DNA segment using molecular biological techniques, such 
as polymerase chain reaction (PCR.TM.) or screening a cDNA or genomic 
library, using primers or probes with sequences based on the above 
nucleotide sequence. The practice of such techniques is a routine matter 
for those of skill in the art, as taught in various scientific articles, 
such as Sambrook et al. (1989), incorporated herein by reference. Certain 
documents further particularly describe suitable mammalian expression 
vectors, e.g., U.S. Pat. No. 5,168,050, incorporated herein by reference. 
Neurotrophic genes and DNA segments that are particularly preferred for 
use in certain aspects of the present methods are the NGF and BDNF genes. 
It is also contemplated that one may clone further genes or cDNAs that 
encode a growth factor or neurotrophic protein or polypeptide. The 
techniques for cloning DNA molecules, i.e., obtaining a specific coding 
sequence from a DNA library that is distinct from other portions of DNA, 
are well known in the art. This can be achieved by, for example, screening 
an appropriate DNA library which relates to the cloning of a CNS healing 
gene. The screening procedure may be based on the hybridization of 
oligonucleotide probes, designed from a consideration of portions of the 
amino acid sequence of known DNA sequences encoding related neurotrophic 
proteins. The operation of such screening protocols are well known to 
those of skill in the art and are described in detail in the scientific 
literature, for example, in Sambrook et al. (1989), incorporated herein by 
reference. 
Techniques for introducing changes in nucleotide sequences that are 
designed to alter the functional properties of the encoded proteins or 
polypeptides are well known in the art, e.g., U.S. Pat. No. 4,518,584, 
incorporated herein by reference, which techniques are also described in 
further detail herein. Such modifications include the deletion, insertion 
or substitution of bases, and thus, changes in the amino acid sequence. 
Changes may be made to increase the neurotrophic activity of a protein, to 
increase its biological stability or half-life, to change its 
glycosylation pattern, and the like. All such modifications to the 
nucleotide sequences are encompassed by this invention. 
It will, of course, be understood that one or more than one neurotrophic 
gene may be used in the methods and compositions of the invention. The 
nucleic acid delivery methods may thus entail the administration of one, 
two, three, or more, neurotrophic genes. The maximum number of genes that 
may be applied is limited only by practical considerations, such as the 
effort involved in simultaneously preparing a large number of gene 
constructs or even the possibility of eliciting an adverse cytotoxic 
effect. The particular combination of genes may be two or more distinct GF 
genes; or it may be such that a GF gene is combined with another 
neurotrophic gene and/or another protein such as a cytoskeletal protein, 
cofactor or other biomolecule; a hormone or growth factor gene may even be 
combined with a gene encoding a cell surface receptor capable of 
interacting with the polypeptide product of the first gene. 
In using multiple genes, they may be combined on a single genetic construct 
under control of one or more promoters, or they may be prepared as 
separate constructs of the same or different types. Thus, an almost 
endless combination of different genes and genetic constructs may be 
employed. Certain gene combinations may be designed to, or their use may 
otherwise result in, achieving synergistic effects on cell stimulation and 
CNS cell growth, any and all such combinations are intended to fall within 
the scope of the present invention. Indeed, many synergistic effects have 
been described in the scientific literature, so that one of ordinary skill 
in the art would readily be able to identify likely synergistic gene 
combinations, or even gene-protein combinations. 
It will also be understood that, if desired, the nucleic segment or gene 
could be administered in combination with further agents, such as, e.g., 
proteins or polypeptides or various pharmaceutically active agents. So 
long as a liposomal-genetic material complex forms part of the 
composition, there is virtually no limit to other components which may 
also be included, given that the additional agents do not cause a 
significant adverse effect upon contact with the target cells or tissues. 
The nucleic acids may thus be delivered along with various other agents as 
required in the particular instance. 
3. Gene Constructs and DNA Segments 
As used herein, the terms "gene" and "DNA segment" are both used to refer 
to a DNA molecule that has been isolated free of total genomic DNA of a 
particular species. Therefore, a gene or DNA segment encoding a 
neurotrophic gene refers to a DNA segment that contains sequences encoding 
a neurotrophic protein, but is isolated away from, or purified free from, 
total genomic DNA of the species from which the DNA is obtained. Included 
within the term "DNA segment", are DNA segments and smaller fragments of 
such segments, and also recombinant vectors, including, for example, 
plasmids, cosmids, phage, retroviruses, adenoviruses, and the like. 
The term "gene" is used for simplicity to refer to a functional protein or 
peptide encoding unit. As will be understood by those in the art, this 
functional term includes both genomic sequences and cDNA sequences. 
"Isolated substantially away from other coding sequences" means that the 
gene of interest, in this case, a neurotrophic gene, forms the significant 
part of the coding region of the DNA segment, and that the DNA segment 
does not contain large portions of naturally-occurring coding DNA, such as 
large chromosomal fragments or other functional genes or cDNA coding 
regions. Of course, this refers to the DNA segment as originally isolated, 
and does not exclude genes or coding regions, such as sequences encoding 
leader peptides or targeting sequences, later added to the segment by the 
hand of man. 
This invention provides novel ways in which to utilize various known 
neurotrophic DNA segments and recombinant vectors. As described above, 
many such vectors are readily available, one particular detailed example 
of a suitable vector for expression in mammalian cells is that described 
in U.S. Pat. No. 5,168,050, incorporated herein by reference. However, 
there is no requirement that a highly purified vector be used, so long as 
the coding segment employed encodes a neurotrophic protein and does not 
include any coding or regulatory sequences that would have an adverse 
effect on CNS cells. Therefore, it will also be understood that useful 
nucleic acid sequences may include additional residues, such as additional 
non-coding sequences flanking either of the 5' or 3' portions of the 
coding region or may include various internal sequences, i.e., introns, 
which are known to occur within genes. 
After identifying an appropriate neurotrophic gene or DNA molecule, it may 
be inserted into any one of the many vectors currently known in the art, 
so that it will direct the expression and production of the neurotrophic 
protein when incorporated into a CNS cell. In a recombinant expression 
vector, the coding portion of the DNA segment is positioned under the 
control of a promoter. The promoter may be in the form of the promoter 
which is naturally associated with a neurotrophic gene, as may be obtained 
by isolating the 5' non-coding sequences located upstream of the coding 
segment or exon, for example, using recombinant cloning and/or PCR.TM. 
technology, in connection with the compositions disclosed herein. 
In other embodiments, it is contemplated that certain advantages will be 
gained by positioning the coding DNA segment under the control of a 
recombinant, or heterologous, promoter. As used herein, a recombinant or 
heterologous promoter is intended to refer to a promoter that is not 
normally associated with a neurotrophic gene in its natural environment. 
Such promoters may include those normally associated with other 
neurotrophic genes, and/or promoters isolated from any other bacterial, 
viral, eukaryotic, or mammalian cell. Naturally, it will be important to 
employ a promoter that effectively directs the expression of the DNA 
segment in CNS cells. 
The use of recombinant promoters to achieve protein expression is generally 
known to those of skill in the art of molecular biology, for example, see 
Sambrook et al. (1989). The promoters employed may be constitutive, or 
inducible, and can be used under the appropriate conditions to direct high 
level or regulated expression of the introduced DNA segment. The currently 
preferred promoters are those such as CMV, RSV LTR, the SV40 promoter 
alone, and the SV40 promoter in combination with the SV40 enhancer. 
4. Liposome-Mediated Transfection 
The therapeutic potential for liposome-mediated gene transfer in the 
injured CNS has been successfully demonstrated using the rodent model. 
Based on existing evidence which shows that the systemic injection of 
cDNA:cationic liposome complexes into animals is non-toxic (Stewart et 
al., 1992), the inventors have developed liposome-mediated gene transfer 
methods which demonstrate the surprising rescue of NF loss following TBI. 
These methods have proven to be superior to those methods of the prior art 
in the transfection of post-mitotic cells. Moreover, the present invention 
has demonstrated that liposome-mediated gene transfer can be used to 
effectively incorporate large gene inserts. Specific tissues and cell 
types may be targeted in vivo by the use of selected promoter-enhancer 
elements that are tissue and cell type specific, administration of the 
plasmid regionally into selected tissue compartments (Holt et al., 1990) 
and coupling a targeting ligand to the liposomal surface (Debs et al., 
1987). 
The present invention provides for a liposomal-mediated system for 
transfecting cDNA of neurotrophins into central nervous system cells, and 
represents the first successful use of cationic liposomes as efficient and 
clinically relevant vectors for the transfer of genes into cells of the 
central nervous system. Efficient transfection of genes may result in 
therapeutic levels of expression of neurotrophins and other proteins which 
could be useful in the treatment of a variety of central nervous system 
pathologies including mechanical injury to the brain and spinal cord, 
stroke, neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's 
disease), organically-mediated psychological disorders (e.g., depression, 
panic disorders) and chronic pain syndromes. 
5. Methods of DNA Transfection 
Technology for introduction of DNA into cells is well-known to those of 
skill in the art. Four general methods for delivering a gene into cells 
have been described: (1) chemical methods (Graham and VanDerEb, 1973); (2) 
physical methods such as microinjection (Capecchi, 1980), electroporation 
(Wong and Neumann, 1982; Fromm et al., 1985) and the gene gun (Yang et 
al., 1990); (3) viral vectors (Clapp, 1993; Danos and Heard, 1992; Eglitis 
and Anderson, 1988); and (4) receptor-mediated mechanisms (Wu et al., 
1991; Curiel et al., 1991; Wagner et al., 1992). 
Chemical and physical methods of DNA transfer are relatively inefficient 
processes and are not applicable to studies in which gene transfer needs 
to occur in a relatively high percentage of cells. Therefore, much effort 
has focused on developing viral vectors for gene transfer and on 
developing new compounds, such as liposomes, that would allow DNA transfer 
at relatively high efficiency. Important clinical disadvantages of viral 
vectors include the possibility of replication-competent virus production, 
immunological reactions and toxicity. 
Liposomes have also been used successfully with a number of cell types that 
are normally resistant to transfection by other procedures including T 
cell suspensions, primary hepatocyte cultures and PC 12 cells (Chang and 
Brenner, 1988; Muller et al., 1990). In addition, liposomes are free of 
the DNA length constraints that are typical of viral-based delivery 
systems. Liposomes have been used effectively to introduce genes, drugs 
(Heath et al., 1986; Storm et al., 1988; Balazsovits et al., 1989), 
radiotherapeutic agents (Pikul et al., 1987), enzymes (Imaizumi et al., 
1990; Imaizumi et al., 1990), viruses (Faller and Baltimore, 1984; Wilson 
et al., 1977; Wilson et al., 1979), transcription factors (Debs et al., 
1990) and allosteric effectors (Nicolau et al., 1979) into a variety of 
cultured cell lines and animals. In addition, several successful clinical 
trails examining the effectiveness of liposome-mediated drug delivery have 
been completed (Lopez-Berestein et al., 1985; Coune, 1988). Furthermore, 
several studies suggest that the use of liposomes is not associated with 
autoimmune responses, toxicity or gonadal localization after systemic 
delivery (Nabel et al., 1992; Mori and Fukatsu, 1992). 
Introduction of the liposome-cDNA transfection complex may be by injection, 
and may be systemic injections into peripheral arteries or veins, 
including the carotid or jugular vessels. Injection may also be directly 
into the central nervous system, either by intraventricular 
administration, or directly into the brain tissue itself. Such injection 
may be facilitated by the use of mini-osmotic pumps for long-duration 
infusion, or an intraparenchymal injection apparatus with ventricular 
cannuli or other intraparenchymal devices. In other embodiments, it may be 
desirable to introduce the liposome-cDNA complex directly into the spinal 
cord or surrounding epidural space. This is particularly important in the 
instance of spinal cord injury. In such cases, either direct injections 
into the spinal cord (intramedullary) or into the subarachnoid space would 
be desirable. In other cases, direct or indirect puncture of the epidural 
space may be desired. In the case of brain injury, or in circumstances 
where introduction of GFs into the brain is desirable, such injection may 
be made into the ventricle, the hippocampus, the cortex, or directly into 
the spinal cord. 
6. Neurofilaments 
Previous histopathologic examination of severe human TBI (Adams et al., 
1983; Gennarelli et al., 1989) and animal cortical impact models (Dixon et 
al., 1991) have pointed to the prevalence of axonal damage as a feature of 
injury pathology. Diffuse axonal injury (DAI) is found in injured cortex 
and is characterized by the histopathologic observation of retraction 
bulbs caused by mechanical forces radiating from the cortical impact site. 
Retraction bulbs are characteristic of post-injury cortex but are rarely 
observed in injured hippocampi (Adams et al., 1983; Dautingy et al., 1988; 
Fineman et al., 1993; Troost et al., 1992). A similar distribution of NF 
protein changes has been observed in the present invention. Loss of NF68 
and NF200 was found in cortical tissue (no hippocampal loss) and was 
predominant in the ipsilateral hemisphere. Thus, the regional distribution 
of TBI-induced NF protein loss was similar to that previously reported for 
axonal damage following TBI in human and animal cortical impact models 
(Adams et al., 1983; Dixon et al., 1991; Gennarelli et al., 1989; Yaghmai 
and Povlishock, 1992). In contrast to earlier qualitative work, these 
studies indicate that the overall effect of TBI on NF68 protein levels 
throughout the cortex is reduction, although site-specific increases of 
NF68 in retraction bulbs have been documented (Yaghmai and Povlishock, 
1992). 
The present invention departs from previous qualitative studies and 
demonstrates the effects of severe TBI on the levels of cytoskeletal 
proteins, which are primary components of axons and dendrites. The axonal 
damage observed following severe TBI is likely to be associated with an 
extensive and persistent loss of key neurofilament proteins. Further, the 
presence of low-MW NF68 breakdown products implicates TBI-induced 
proteolysis in the loss of NF protein. 
A surprising aspect of the present invention is the presence of low MW 
immunopositive bands in injured samples associated with loss of the parent 
NF68 protein. These bands do not occur substantially in naive or 
sham-injured samples but appear prominently in 3 h, 1 day, and 7 days 
post-injury samples. These bands may represent NF68 breakdown products 
(BDPs) produced by the action of proteases on the parent NF68 protein, 
since prominent BDPs at MWs of 56 kDa and 52 kDa were observed in Western 
analyses. Interestingly, the action of the calcium-activated protease, 
calpain, on neurofilament protein produces immunopositive cleavage 
products of 57 kDa and 53 kDa. These NF68 BDPs have been observed in both 
in vivo and in vitro studies (Kamakura et al., 1985; Schlaepfer et al., 
1984; Schlaepfer and Zimmerman, 1985a, 1985b). The fragments correspond to 
the amino terminal, alpha-helical domain common to all neurofilament 
proteins (NF68, NF150, NF200). Therefore, NF68 BDPs identified here may be 
fragments produced by cleavage of NF68, NF150, and/or NF200 (Schlaepfer et 
al., 1984; Schlaepfer and Zimmerman, 1985a, 1985b). Since the existing 
anti-NF200 monoclonal antibodies target the carboxyl terminal domain 
present in NF200 (and consequently do not recognize the low molecular 
weight BDPs containing the alpha-helical domain), other antibodies are 
required to determine the relative contribution of NF68, NF150, or NF200 
to the low MW BDPs. 
Although other proteases also produce immunopositive fragments, such as 
cathepsins B and D, trypsin, and alphachymotrypsin, the MWs of the 
proteolytic fragments were substantially lower and did not resemble the 
pattern observed in post-TBI (Chin et al., 1983; Kamakura et al., 1985; 
Nixon and Marotta, 1984). The BDPs observed in this study match very 
closely those produced by calpain-dependent proteolysis and suggest that 
calpain activation contributes to the loss of neurofilament protein 
observed post-injury. These breakdown products are of particular interest 
because they are believed to be involved in the maintenance of 
neurofilament metabolism (Schlaepfer et al., 1984; Schlaepfer and 
Zimmerman, 1985a, 1985b). Thus, the action of TBI may produce alterations 
of neurofilament function by two mechanisms: (1) by reduction of NF68 and 
NF200 protein levels and (2) by production of metabolically significant 
breakdown products. 
Like other neuropathologic conditions, such as ischemia and protracted 
seizures, TBI pathology is produced, at least in part, by prolonged 
excitotoxicity and subsequent calcium influx, leading to a loss of 
intracellular calcium homeostasis. Under more severe TBI magnitudes, such 
as those experienced in cortical impact models, excitotoxic injury is 
accompanied by the presence of mechanical damage. Mechanical forces caused 
by injury can disrupt membrane integrity and, thereby, further contribute 
to intracellular calcium levels. The cascade of biochemical pathologic 
conditions that may occur under conditions of high intracellular calcium 
would include activation of calciumdependent proteases (Siman and Noszek, 
1988). Significantly, lateral fluid percussion injury (a model of TBI that 
produces severe injury) causes higher levels of intracellular calcium in 
injured cortex than in the corresponding hippocampi (Fineman et al., 
1993). These findings may explain the distribution of calpain-like BDPs in 
the cortex, where injury and calcium accumulation may be most severe, and 
its notable absence in the hippocampus. 
An important aspect of the present invention is the use of two models of 
TBI which reproduce features of moderate diffuse TBI or more severe injury 
with focal tissue damage. This approach permits the study of 
liposome-mediated gene transfection across a clinically-relevant range of 
injury levels. Fluid percussion injury, a model of diffuse TBI, produces 
deficits in motor function and spatial memory performance in part by 
producing sublethal excitotoxic damage of neurons (Hayes et al., 1992). 
The hippocampus is preferentially vulnerable in this model (Lyeth et al., 
1990). Several studies have documented that fluid percussion TBI does not 
produce cerebral ischemia (DeWitt et al., 1989), but sustained blood-brain 
barrier opening is observed (Jiang et al., 1992). Cortical impact, a model 
of severe TBI, produces focal contusions and more DAI than fluid 
percussion injury (Dixon et al., 1991). Excitotoxic injury processes also 
contribute to enduring behavioral deficits following cortical impact 
(Palmer et al., 1993). 
A particular aspect of the present invention is the evidence provided which 
suggests NF proteolysis plays an important role in pre-necrotic axonal 
changes after TBI. The loss of NF68 and NF200 proteins, as well as the 
appearance of NF68 BDPs, strongly suggests the occurrence of pathologic 
proteolysis post-injury. Data describing NF protein loss secondary to 
calpain activation in models of spinal cord injury further support the 
protease hypothesis (Banik et al., 1992; Schlaepfer and Zimmerman, 1985a). 
Importantly, the observation of immunopositive NF68 BDPs in the cortical 
impact model complicates the interpretation of immunohistochemical data 
and could provide an alternate explanation for localized increases in NF68 
immunoreactivity post-injury (Povlishock, 1993; Yaghmai and Povlishock, 
1992). Increased NF68 antigenicity in immunohistochemical studies may 
represent BDPs of NF68, as well as NF150 and NF200. 
In laboratory models of ischemia, NF protein loss has been demonstrated 
after focal ischemic damage (Inuzuka et al., 1990b), as well as after 
global ischemia in both rats (Kaku et al., 1993) and gerbils (Kamakura et 
al., 1985; Nakamura et al., 1992). Similarly, cytoskeletal damage caused 
by ischemia has been prevented by administration of antiproteolytic 
compounds (Arai et al., 1990, 1991; Inuzuka et al., 1990a; Lee et al., 
1991). 
Two related hypotheses have been proposed that address the mechanism of 
axonal damage and diffuse axonal injury following TBI (Balentine, 1985; 
Povlishock, 1993). The first postulate implicates primary disruption of 
NF, possibly associated with altered NF phosphorylation, as contributing 
to NF disassembly and localized increases in NF antigenicity found in 
retraction balls (Povlishock, 1993; Yaghmai and Povlishock, 1992). To 
date, no direct evidence exists for altered phosphorylation of NF after 
TBI. The second premise suggests that TBI activates calciumdependent 
proteases that cause subsequent NF disruption and proteolysis. 
7. Traumatic Brain Injury 
Axonal injury has long been thought to be a primary pathologic feature of 
traumatic brain injury (TBI). Animal models of severe TBI and human 
post-injury pathology demonstrate axonal damage (Gennarelli et al., 
Yaghmai and Povlishock, 1992). A central polemic in TBI investigation is 
the relative roles of structural determinants vs. concurrent biochemical 
derangements as the crucial pathologic events post-injury. In human 
studies, investigators have identified diffuse axonal injury (DAI) as the 
key pathologic feature responsible for the neurobehavioral deficits that 
accompany human TBI (Adams et al., 1983; Gennarelli et al., 1989). 
Current studies investigating DAI after TBI have been predominantly 
pathomorphologic and qualitative in nature. Recent investigations of the 
role of NF68 in DAI pathology are a noteworthy exception (Yaghmai and 
Povlishock, 1992). Although axonal dysfunction may play an important role 
in TBI pathology, no causal relationship has been established between DAI 
and post-injury neurobehavioral deficits. In fact, numerous studies have 
revealed that functionally normal neurotransmission can be preserved in 
pathways that have suffered significant loss of fibers (Beattie et al., 
1988; Sautter et al., 1991). Thus, a comprehensive quantitative assessment 
of cytoskeletal alterations would contribute to the understanding of DAI 
and its role in TBI pathology. 
Rodent models of TBI cause neurobehavioral deficits, including motor and 
memory disturbances, that resemble those seen in human patients (Hamm et 
al., 1992; Lyeth et al., 1990). Little is known of the molecular events 
leading to post-injury pathology in these animal models. Recent laboratory 
studies have determined that TBI induces a significant decrease in the 
protein levels of key dendritic cytoskeletal elements (Taft et al., 1992, 
1993), including microtubule-associated protein 2 (MAP2). Post-TBI 
excitotoxicity (Gorman et al., 1989; Hayes et al., 1992), loss of calcium 
homeostasis (Fineman et al., 1993) and pathologic activation of 
calcium-dependent proteases (Taft et al., 1992, 1993) (e.g., calpain) may 
be principal causes of cytoskeletal degradation. Although calpains are 
found in both axonal and dendritic environments (Perlmutter et al., 1988), 
previous examination of cytoskeletal pathology after TBI has focused on 
dendritic cytoskeletal elements (MAP2) (Taft et al., 1992, 1993). 
Neurofilament proteins (Kamakura et al., 1985; Schlaepfer et al., 1984; 
Schlaepfer and Zimmerman, 1985b), MAP2 (Johnson et al., 1991), spectrin 
(Siman et al., 1984), and other cytoskeletal proteins are substrates for 
calpain-dependent proteolysis. 
Therapeutically relevant strategies for manipulating production of these 
proteins may ultimately have important implications for the treatment of 
TBI. TBI is associated with disturbances of the blood-brain barrier which 
may enhance delivery of transgenes by vectors that would otherwise have 
restricted access to the CNS. In contrast to neurodegenerative diseases, 
experimental TBI can produce relatively rapid changes in specific 
proteins, facilitating assessments of the effects of gene therapy on 
post-traumatic gene expression. 
8. Neuronal Architecture 
Neurofilament (NF) proteins are primary components of the neuronal 
cytoarchitecture, including axons and dendrites (Shaw, 1986). NFs consist 
of three separate protein elements collectively called the neurofilament 
triplet proteins (Hoffman and Lasek, 1975). These subunits have apparent 
molecular weights of 200 kDa (NF-H), 150 kDa (NF-M), and 68 kDa (NF-L) as 
estimated by gel electrophoresis (Dautingy et al., 1988). The 68-kDa 
subunit is an assembly protein found predominantly in the neurofilament 
core, and the 150-kDa and 200-kDa subunits are cross-linking proteins 
found in the connecting branches. 
All NF triplet proteins exhibit significant sequence homology in the amino 
terminal, alpha helical domain. The 150-kDa and 200-kDa subunits possess 
highly repeating lysine-serine-proline (KSP) sequences, which are heavily 
phosphorylated and contribute to anomalous electrophoretic mobilities 
(Julien and Mushynski, 1982; Nixon and Sihang, 1991). 
Neurofilament rescue refers to the prevention of neurofilament 
deterioration, and/or to the maintenance and/or promotion of neurofilament 
restoration and recovery. Such rescue may be accomplished by reducing the 
rate of neurofilament decrease following TBI or by enhancing and/or 
stimulating the rate of neurofilament protein production and nerve cell 
restoration. 
The assembly of NF proteins results in the formation of neurofilaments of 
approximately 10 nm in diameter in the axon hillock that are transported 
down the axon. Although their function is primarily structural, NF 
proteins have been implicated in many disease processes, such as 
Alzheimer's disease (Sternberger et al., 1985; Ulrich et al., 1987), 
amyotrophic lateral sclerosis (Troost et al., 1982), and Pick's disease 
(Pietrini et al., 1993), and in neurologic insults, such as stroke (Arai 
et al., 1990, 19910; Inuzuka et al., 1990a, b; Kaku et al., 1993; Kudo et 
al., 1993; Lee et al., 1991; Nakamura et al., 1992).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The inventors have employed primary septo-hippocampal cell cultures to 
examine liposomal-mediated gene transfection in vitro, and have determined 
optimal ratios for transfection employing the .beta.-galactosidase 
reporter gene. Transfection efficiency was significantly improved over the 
prior art results using liposomal-mediated systems in vitro (Kaech et al., 
1993). RT-PCR analyses have confirmed increased expression of BDNF and 
NGF. Initial in situ hybridization analyses further confirmed successful 
transfection of BDNF cDNA in vitro. ELISA analyses have detected large 
increases in NGF protein after in vitro liposomal transfection. .beta.-Gal 
has been used to study transfection following local injections of 
cDNA-liposome complexes in the region of the hippocampus. These studies 
also indicate successful liposomal mediated transfection in uninjured 
rats. In order to accurately assess the effects of gene transfection 
following trauma, in situ hybridization studies have been performed to 
characterize changes in endogenous NGF and BDNF after injury. 
Liposome-mediated gene transfection of nerve growth factor (NGF) has been 
exploited in primary central nervous system cultures. RT-PCR analyses 
detected increased expression of NGF mRNA one day after liposome-mediated 
NGF gene transfection; ELISA studies detected large increases in NGF 
protein in cells and in culture medium after NGF gene transfection. Cells 
continued to secrete NGF into the medium for at least 2 weeks. NGF 
bioassays confirmed that the NGF secreted after gene transfection was 
biologically active. 
In situ hybridization and immunohistochemistry have demonstrated in the 
present invention the successful expression of NGF and BDNF mRNA at 
various times after administration of cDNA-liposome complexes. Protein 
expression analyses as well as immunohistochemical analyses of 
neurotrophin receptors (p75.sup.NGFR, p140.sup.trkB) and bioactivity assay 
of NGF and BDNF have also demonstrated the presence of these growth 
factors in vitro and in vivo. Gene therapy has been employed to achieve 
therapeutically useful levels of expression of neurotrophins and other 
proteins in the traumatically injured brain. Experimental models of TBI 
have been used to facilitate the study of transfection of central nervous 
system cells. 
1. Forty-fold Improvement in Transfection Efficiency 
Studies employing the .beta.-Gal reporter gene in CNS cell cultures further 
confirmed observations of other researchers that the concentrations of 
liposomes can influence the efficiency of transfection (Felgner et al., 
1987). The differential transfection efficiencies in CNS cell cultures 
associated with varying concentrations of liposomes is consistent with the 
view that the higher the net positive charge of DNA-liposome complexes is, 
the better the interaction with the negatively charged cell membrane will 
be. However, overly high levels of liposomes can cause cell lysis. Without 
increasing liposome concentrations, increased amounts of DNA did not 
improve transfection efficiency. 
The efficiency of the DOTMA and DOPE mediated pCMV/.beta.-Gal transfection 
observed by the inventors in septo-hippocampal cell cultures (&gt;1000 
transfected cells per 16 mm well) exceeds by nearly forty-fold the 
previously reported transfection efficiency for .beta.-Gal in hippocampal 
cultures employing the transfection reagents Transfectam and DOTAP (40-200 
per 35mm well) (Kaech et al., 1993). The sustained expression of 
.beta.-Gal for at least two weeks further suggests the potential 
therapeutic utility of liposomal mediated gene transfection in CNS injury 
and degeneration. 
2. 1:3 is Optimum Ratio of cDNA:Liposomes for Transfection 
.beta.-Gal has been used as a reporter gene for examining factors 
influencing the efficiency of liposome-mediated gene transfection in 
central nervous system cell cultures. Results indicate that without 
increasing the amounts of DNA, increased liposome concentrations within 
certain limits enhanced transfection efficiency. However, higher liposome 
levels could produce cell lysis. Without increasing liposome 
concentrations, increased amounts of DNA did not improve transfection 
efficiency. Employing the optimal concentration (1 .mu.g DNA/3 .mu.l 
liposomes/well), .beta.-Gal gene expression was sustained for at least two 
weeks after transfection in primary septo-hippocampal cultures. 
Studies of liposome mediated gene transfection in septo-hippocampal 
cultures determined that a concentration of 1 .mu.g DNA/3 .mu.l 
liposomes/well (16-mm well) (or a ratio of 1:3) produced superior results, 
and this ratio was used to transfer the NGF gene to primary 
septo-hippocampal cell cultures. The purpose of these studies was to 
systematically examine if liposome-mediated NGF gene transfection could 
produce increased expression of NGF mRNA and protein. The biological 
activity of the NGF protein produced following transfection was also 
analyzed. Ratios of cDNA to liposomes were tested in the range of about 
1:1 to about 1:9. Ratios of about 1:1 to about 1:5 were particularly 
preferred, with a ratio of about 1:3 being most preferred. 
3. Successful Neurotrophin Transfection in Vitro and in Vivo 
Gene transfection could result in increased expression of trophic proteins 
or decreased expression of toxic proteins. Routes of administration of 
liposomal-cDNA complexes could include direct delivery into the central 
nervous system (local, ventricular, and/or epidural injections) or 
systemic injections. Specific tissues and cell types could be targeted by 
several approaches. These include using promoter enhancer elements that 
are tissue and cell type specific, administration of the plasma regionally 
into selected tissue compartments as indicated above and coupling a 
targeting ligand to the liposomal surface. 
4. BDNF and NGF Transfection Rescues Neurofilament Loss After TBI 
A post-injury analysis of neurofilament proteins in the cortex and 
hippocampal tissue was performed. Lateral cortical impact injury resulting 
in severe TBI causes pronounced reduction in NF68 and NF200 levels that 
lasts for at least 2 weeks post-injury. Further, the presence of low 
molecular weight (MW) NF68 immunopositive bands of 52 kDa and 56 kDa may 
indicate the involvement of calpain-mediated proteolysis. The temporal and 
regional profiles of these changes could have profound implications for 
axonal and dendritic pathology after severe TBI. 
Primary septo-hippocampal cell cultures have been employed to demonstrate 
the therapeutic potential of BDNF gene transfection in facilitating the 
recovery of neurofilament loss caused by depolarization injury. Employing 
a pUC19-based plasmid, rat BDNF cDNA was subcloned into a unique NotI site 
under the control of the CMV promoter to generate pBDNF. DNA for BDNF was 
complexed with liposomes and transfected into primary septo-hippocampal 
cell cultures one day after depolarization injury (6.0 min depolarization 
with 60 mM KCl and the presence of 2.8 mM Ca.sup.++). Three days after 
depolarization injury, Western blot and immunohistochemical analyses 
detected significant loss (42%) of NF-M and NF-H proteins (Sternberger SMI 
31 antibody) in untreated cultures. However, densitometric scanning of 
Western blot data indicated that BDNF transfection produced a two-fold 
increase in NF-M/NF-H three days following injury as compared to untreated 
cultures. Immunohistochemical studies also detected enhanced NF-M/NF-H 
immunolabeling in injured neurons following BDNF transfection as compared 
to untransfected, injured controls. Thus, BDNF gene transfection could be 
a useful therapeutic tool for blunting neurofilament loss associated with 
injury to central nervous system neurons. 
5. Use of the Cytomegalovirus Promoter 
When the gene remains extrachromosomal, as in liposomal mediated gene 
transfections, optimal levels of expression are likely to be achieved 
using viral promoters. Furthermore, viral promoters generally function in 
a broad range of cell types. The relative strengths of several commonly 
used viral promoters have been studied in different cell lines including 
primary cultures of rat mammary epithelial cells, NIH 3T3 (Thompson et 
al., 1993), primary rat and human hepatocytes (Fang et al., 1989; Li et 
al., 1992), human embryo fibroblasts (Giordano et al., 1991) and brain 
tumor cell lines (Dellig and Seliger, 1990). Studies indicate that the 
cytomegalovirus is a prudent initial choice of viral promoter. In primary 
cultures of human hepatocytes, the CMV promoter yields a higher 
transfection efficiency than RSV and SV40 (Li et al., 1992). RSV and CMV 
promoters were tested for activity are substantial in non-replicating 
cells: the efficiency of RSV promoter is not greater than background 
whereas the CMV promoter is very active. Furthermore, the CMV promoter 
exhibits two-fold greater activity in growing cells (Giordano et al., 
1991). The CMV promoter also shows high activity in glioblastoma cell 
lines (Dellig and Seliger, 1990). 
6. Immunocytochemistry of NGF and BDNF 
RT-PCR and in situ hybridization techniques may be employed to determine 
the transfection efficiencies for NGF and BDNF. Autoradiographic and 
emulsion in situ hybridization techniques employ .sup.33 P-labeled mRNA 
probes for NGF and BDNF to provide information on the regional extent, 
cellular localization and persistence of increased mRNA. RT-PCR methods 
provide semiquantitative information on changes in mRNA. RT-PCR techniques 
are considerably more sensitive than Northern blots and are more robust to 
the effects of injury. RT-PCR has been reliably used to evaluate levels of 
NGF and BDNF mRNA in rodent brains (Giordano et al., 1992). In situ 
hybridization studies are important for development of appropriate 
dissection protocols for TR-PCR analyses in vivo, since, in vivo 
transfection will be restricted to specific areas of the brain. 
A monoclonal antibody (clone 27/21) specific for rat and mouse .beta. 
(2.5S) NGF that is also suitable for ELISA determinations of NGF proteins 
in brain tissue was used for protein assessment. An antibody for BDNF 
having considerable specificity has been reported (Denton et al., 1993), 
and Dr. Franz Hafti of Genentech, Inc. provided this antibody. 
Immunohistochemistry may be employed to describe regional changes in 
expression of NGF and BDNF protein. ELISA may be used to provide 
quantitative data on changes in neurotrophin protein levels. 
Immunohistochemical analyses of neurotrophin receptors p75.sup.NGFR, 
p140.sup.trkA, p145.sup.trkB may be performed. Antibodies are made to 
synthetic peptide segments on the different receptors and allow 
determination of different tyrosine kinase receptors (TRKs) as well as 
differential staining of full and truncated versions of the different 
TRKs. Neurotrophins bind with equal affinity to a common low affinity 
neurotrophin receptor, p75.sup.NGFR whose function is likely to be 
stimulation of high affinity binding to trk receptors (Chao et al., 1993, 
Battleman et al., 1993). 
The different neurotrophins also bind to different members of the trk 
family of membrane-spanning tyrosine kinase receptors, the signal 
transducing elements of the neurotrophins (Barker and Murphy, 1992). NGF 
binds specifically to p140.sup.trkA. BDNF binds specifically to 
p145.sup.trkB. NT-3 binds to both p145.sup.trkB and p145.sup.trkC. 
Although there are claims that p140.sup.trkA by itself has high affinity 
NGF-binding properties, others report that both genes are required for 
high affinity NGF-binding activity (Chao et al., 1993, Battleman et al., 
1993). 
7. Biological Functional Equivalents 
As mentioned above, modification and changes may be made in the structure 
of a neurotrophic gene and still obtain a functional molecule that encodes 
a protein or polypeptide with desirable characteristics. The following is 
a discussion based upon changing the amino acids of a protein to create an 
equivalent, or even an improved, second-generation molecule. The amino 
acid changes may be achieved by changing the codons of the DNA sequence, 
according to the following data (Table 1) 
TABLE 1 
______________________________________ 
Amino Acids Codons 
______________________________________ 
Alanine Ala A GCA GCC GCG GCU 
Cysteine Cys C UGC UGU 
Aspartic acid 
Asp D GAC GAU 
Glutamic acid 
Glu E GAA GAG 
Phenylalanine 
Phe F UUC UUU 
Glycine Gly G GGA GGC GGG GGU 
Histidine 
His H CAC CAU 
Isoleucine 
Ile I AUA AUC AUU 
Lysine Lys K AAA AAG 
Leucine Leu L UUA UUG CUA CUC CUG CUU 
Methionine 
Met M AUG 
Asparagine 
Asn N AAC AAU 
Proline Pro P CCA CCC CCG CCU 
Glutamine 
Gln Q CAA CAG 
Arginine Arg R AGA AGG CGA CGC CGG CGU 
Serine Ser S AGC AGU UCA UCC UCG UCU 
Threonine 
Thr T ACA ACC ACG ACU 
Valine Val V GUA GUC GUG GUU 
Tryptophan 
Trp W UGG 
Tyrosine Tyr Y UAC UAU 
______________________________________ 
For example, certain amino acids may be substituted for other amino acids 
in a protein structure without appreciable loss of interactive binding 
capacity with structures such as, for example, antigen-binding regions of 
antibodies or binding sites on substrate molecules. Since it is the 
interactive capacity and nature of a protein that defines that protein's 
biological functional activity, certain amino acid sequence substitutions 
can be made in a protein sequence, and, of course, its underlying DNA 
coding sequence, and nevertheless obtain a protein with like properties. 
It is thus contemplated by the inventors that various changes may be made 
in the DNA sequences of neurotrophic genes without appreciable loss of 
their biological utility or activity. 
In making such changes, the hydropathic index of amino acids may be 
considered. The importance of the hydropathic amino acid index in 
conferring interactive biologic function on a protein is generally 
understood in the art (Kyte & Doolittle, 1982, incorporate herein by 
reference). It is accepted that the relative hydropathic character of the 
amino acid contributes to the secondary structure of the resultant 
protein, which in turn defines the interaction of the protein with other 
molecules, for example, enzymes, substrates, receptors, DNA, antibodies, 
antigens, and the like. 
Each amino acid has been assigned a hydropathic index on the basis of their 
hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these 
are: Isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine 
(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); 
glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); 
tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); 
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and 
arginine (-4.5). 
It is known in the art that certain amino acids may be substituted by other 
amino acids having a similar hydropathic index or score and still result 
in a protein with similar biological activity, i.e., still obtain a 
biological functionally equivalent protein. In making such changes, the 
substitution of amino acids whose hydropathic indices are within .+-.2 is 
preferred, those which are within .+-.1 are particularly preferred, and 
those within .+-.0.5 are even more particularly preferred. 
It is also understood in the art that the substitution of like amino acids 
can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 
4,554,101, incorporated herein by reference, states that the greatest 
local average hydrophilicity of a protein, as governed by the 
hydrophilicity of its adjacent amino acids, correlates with a biological 
property of the protein. 
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values 
have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); 
aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine 
(+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline 
(-0.5.+-.1); alanine (-0.5); histidine *-0.5); cysteine (-1.0); methionine 
(-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); 
phenylalanine (-2.5); tryptophan (-3.4). 
It is understood that an amino acid can be substituted for another having a 
similar hydrophilicity value and still obtain a biologically equivalent, 
and in particular, an immunologically equivalent protein. In such changes, 
the substitution of amino acids whose hydrophilicity values are within +2 
is preferred, those which are within +1 are particularly preferred, and 
those within .+-.0.5 are even more particularly preferred. 
As outline above, amino acid substitutions are generally therefore based on 
the relative similarity of the amino acid side-chain substituents, for 
example, their hydrophobicity, hydrophilicity, charge, size, and the like. 
Exemplary substitutions which take various of the foregoing 
characteristics into consideration are well known to those of skill in the 
art and include: arginine and lysine; glutamate and aspartate; serine and 
threonine; glutamine and asparagine; and valine, leucine and isoleucine. 
8. Site-Specific Mutagenesis 
Site-specific mutagenesis is a technique useful in the preparation of 
individual peptides, or biologically functional equivalent proteins or 
peptides, through specific mutagenesis of the underlying DNA. The 
technique further provides a ready ability to prepare and test sequence 
variants, for example, incorporating one or more of the foregoing 
considerations, by introducing one or more nucleotide sequence changes 
into the DNA. Site-specific mutagenesis allows the production of mutants 
through the use of specific oligonucleotide sequences which encode the DNA 
sequence of the desired mutation, as well as a sufficient number of 
adjacent nucleotides, to provide a primer sequence of sufficient size and 
sequence complexity to form a stable duplex on both sides of the deletion 
junction being traversed. Typically, a primer of about 17 to 25 
nucleotides in length is preferred, with about 5 to 10 residues on both 
sides of the junction of the sequence being altered. 
In general, the technique of site-specific mutagenesis is well known in the 
art, as exemplified by various publications. As will be appreciated, the 
technique typically employs a phage vector which exists in both a single 
stranded and double stranded form. Typical vectors useful in site-directed 
mutagenesis include vectors such as the M13 phage. These phage are readily 
commercially available and their use is generally well known to those 
skilled in the art. Double stranded plasmids are also routinely employed 
in site directed mutagenesis which eliminates the step of transferring the 
gene of interest from a plasmid to a phage. 
In general, site-directed mutagenesis in accordance herewith is performed 
by first obtaining a single-stranded vector or melting apart of two 
strands of a double stranded vector which includes within its sequence a 
DNA sequence which encodes the desired neurotrophic protein. An 
oligonucleotide primer bearing the desired mutated sequence is prepared, 
generally synthetically. This primer is then annealed with the 
single-stranded vector, and subjected to DNA polymerizing enzymes such as 
E. coli polymerase I Klenow fragment, in order to complete the synthesis 
of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one 
strand encodes the original non-mutated sequence and the second strand 
bears the desired mutation. This heteroduplex vector is then used to 
transform appropriate cells, such as E. coli cells, and clones are 
selected which include recombinant vectors bearing the mutated sequence 
arrangement. 
The preparation of sequence variants of the selected neurotrophic gene 
using site-directed mutagenesis is provided as a means of producing 
potentially useful species and is not meant to be limiting as there are 
other ways in which sequence variants of neurotrophic genes may be 
obtained. For example, recombinant vectors encoding the desired 
neurotrophic gene may be treated with mutagenic agents, such as 
hydroxylamine, to obtain sequence variants. 
In certain embodiments, it will be advantageous to employ nucleic acid 
sequences of the present invention in combination with an appropriate 
means, such as a label, for determining hybridization. A wide variety of 
appropriate indicator means are known in the art, including fluorescent, 
radioactive, enzymatic or other ligands, such as avidin/biotin, which are 
capable of giving a detectable signal. In preferred embodiments, one will 
likely desire to employ a fluorescent label or an enzyme tag, such as 
urease, alkaline phosphatase or peroxidase, instead of radioactive or 
other environmental undesirable reagents. In the case of enzyme tags, 
calorimetric indicator substrates are known that can be employed to 
provide a means visible to the human eye or spectrophotometrically, to 
identify specific hybridization with complementary nucleic acid-containing 
samples. 
In general, it is envisioned that the hybridization probes described herein 
will be useful both as reagents in solution hybridization as well as in 
embodiments employing a solid phase. In embodiments involving a solid 
phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a 
selected matrix or surface. This fixed, single-stranded nucleic acid is 
then subjected to specific hybridization with selected probes under 
desired conditions. The selected conditions will depend on the particular 
circumstances based on the particular criteria required (depending, for 
example, on the G+C content, type of target nucleic acid, source of 
nucleic acid, size of hybridization probe, etc.). Following washing of the 
hybridized surface so as to remove nonspecifically bound probe molecules, 
specific hybridization is detected, or even quantitated, by means of the 
label. 
The following examples are included to demonstrate preferred embodiments of 
the invention. It should be appreciated by those of skill in the art that 
the techniques disclosed in the examples which follow represent techniques 
discovered by the inventors to function well in the practice of the 
invention, and thus can be considered to constitute preferred modes for 
its practice. However, those of skill in the art should, in light of the 
present disclosure, appreciate that many changes can be made in the 
specific embodiments which are disclosed and still obtain a like or 
similar result without departing from the spirit and scope of the 
invention. 
EXAMPLE 1 
OPTIMIZING IN VITRO TRANSFECTION EFFICIENCY USING .beta.-GAL 
Previous work has shown that the ratio of nucleic acid to liposomes during 
transfection is critical for optimizing transfection efficiency in 
cultured cells. 1 .mu.g of plasmid DNA in 100 .mu.l Dulbecco's Modified 
Eagle medium (D-MEM) was mixed with 1 .mu.l or 3 .mu.l of cationic 
liposomes diluted in 100 .mu.l D-MEM, and overlaid overnight onto rat 
hippocampal cells, 80 to 90% confluent (2.8.times.10.sup.5 cells per 15 mm 
plate). X-Gal staining was performed 48 hours after transfection to 
calculate transfection efficiency. Employing a 1:3 (DNA/liposome) 
transfection ratio, X-Gal staining was detected both in cells having 
neuronal and astrocytic morphology (FIG. 1A). 
Less efficient transfection was observed with a 1:1 transfection ratio 
(FIG. 1B vs. FIG. 1C). Employing a 1:3 ratio of cDNA to liposomes, 
.beta.-Gal transfection efficiency was calculated from X-Gal staining and 
septo-hippocampal cultures at three time points after transfection with 
cDNA for the reporter gene, .beta.-Gal (FIG. 2). Maximal transfection 
(&gt;3%) was detected two days after injury and persisted for at least one 
week. 
The efficiency of transfection of .beta.-Gal observed in cultured 
hippocampal neurons (&gt;3%) far exceeds previously reported transfection 
efficiency for .beta.-Gal in hippocampal cultures employing transfection 
reagents Transfectam and DOTAP (&lt;0.02%) (Kaech et al., 1993). The 
differential efficiencies associated with varying ratios of cDNA to 
liposomes is consistent with the view that the higher the net positive 
charge of DNA-liposomes complexes, the better the interaction with a 
negatively charged cell membrane. 
EXAMPLE 2 
IN VITRO NGF AND BDNF TRANSFECTION 
Employing transfection procedures described herein (see Example 1), 
RT-PCR.TM. analyses were performed using mRNA for BDNF one day and one 
week after transfection of septo-hippocampal cultures employing varying 
cDNA to liposome ratios (FIG. 3A, FIG. 3B, and FIG. 3C). Sham 
transfections employing only liposomes were also included. Total RNA 
isolated from each well was used for reverse transcription for cDNA. To 
check for possible DNA contamination during RNA preparation, the same RNA 
samples were included without performing the reverse transcription 
procedure. RT-PCR confirmed increased message for BDNF for all ratios 
tested, as compared to sham transfections. Control studies confirmed the 
absence of DNA contamination. 
FIG. 3B shows that 1:4 and 2:3 ratios produced higher levels of mRNA for 
BDNF one week following transfection than produced by the 1:3 ratio. The 
inventors also observed increased levels in endogenous BDNF in vitro seen 
at 1 week in sham transfected preparations (FIG. 3B) and compared to 1 day 
after sham transfection (FIG. 3A). RT-PCR analyses also confirmed 
increased mRNA for NGF one day after transfection (FIG. 3C). Preliminary 
in situ analyses employing .sup.33 P-labeled RNA probe for BDNF also 
detected prominent radiolabeling (FIG. 4A) superimposed over cell bodies 
stained with hematoxylin (FIG. 4B). 
ELISA studies have detected dramatic increases in NGF protein both in cells 
and media 3 days after transfection of septo-hippocampal cultures (FIG. 
5). These large increases in NGF indicate that transfected cells are not 
only producing NGF but also releasing the protein into the culture medium. 
The observation of similar profiles of neurotrophin production in vivo 
would strongly suggest that transfection of neurotrophin genes would have 
therapeutic potential in the intact animal. 
EXAMPLE 3 
.beta.-GAL TRANSFECTION EFFICIENCY IN VIVO USING UNINJURED RATS 
As shown in FIG. 6, the present invention provides evidence that direct 
intracerebral injections of cDNA:liposomal complexes (1:3 ratio) into the 
dorsal hippocampus of uninjured rats can produce transfection of the 
.beta.-Gal reporter gene four days after injection. Initial microscopic 
analyses indicate X-Gal staining in cells having apparent neuronal and 
glial morphology. 
EXAMPLE 4 
NGF AND BDNF EXPRESSION IN SHAM INJURED AND INJURED RATS 
In studies of transfection of BDNF and NGF following TBI, it is important 
to describe the effects of injury on expression of endogenous BDNF and 
NGF. In situ hybridization analyses of expression of endogenous BDNF have 
been performed following sham injury (FIG. 7A), 3 hours after cortical 
impact injury (FIG. 7B), or 1 day following cortical impact injury (FIG. 
7C). Cortical impact injury produced prominent increases in expression of 
endogenous BDNF, especially in the region of the dorsal hippocampus, as 
compared to sham injury controls. By 1 day following injury, expression of 
endogenous BDNF had returned approximately to control levels. Emulsion in 
situ hybridization analyses have been utilized to provide detail on the 
cellular localization of changes in expression of endogenous BDNF three 
hours following cortical impact injury (FIG. 8A and FIG. 8B). These 
studies detected increased radiolabeling primarily localized over cell 
bodies in regions such as the dentate gyrus and CA4 of the hippocampus. 
EXAMPLE 5 
CONSTRUCTION OF VECTORS AND LIPOSOME FORMULATION 
The cytomegalovirus (CMV) promoter (Fang et al., 1989; Giordano et al., 
1991; Li et al., 1992; Thompson et al., 1993) was incorporated into a 
pUC19-derived expression vector for construction of the NGF and BDNF 
transfection vectors (pNGF and PBDNF, respectively) (MacGregor and Caskey, 
1989; Yang et al., 1994a). The rat NGF and BDNF cDNAs were subcloned 
individually into a unique NotI site under the control of the CMV 
promoter. The BDNF and NGF genes were obtained from C. Y. Hsu, Department 
of Neurology, Washington University School of Medicine, St. Louis, Mo. 
A commercially available, 1:1 (w:w) mixture of the cationic lipid 
n-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 
dioleoyl phosphotidylethanolamine (DOPE) was used for the liposome 
formulation and was prepared in membrane-filtered water (GIBCO-BRL, 
Bethesda, Md.). 
EXAMPLE 6 
EXPRESSION OF mRNA FOLLOWING TRANSFECTION 
1. RT-PCR analyses of NGF and BDNF mRNA 
1 day after liposome-mediated NGF or BDNF gene transfection (1 .mu.g DNA/3 
.mu.l liposomes/well), cells in culture were lysed by adding 0.2 ml of 
RNAzol B (Cinns/Biotec Laboratories, Inc.) per 10.sup.6 cells. The RNA 
preparations were performed as previously described (Yang et al., 1993). 
Total RNA from individual wells was used for DNA synthesis at 42.degree. 
C. for 2 h using 200 units of M-MLV reverse transcriptase (Perkin-Elmer, 
Norwalk, Conn.), 40 nmol of dNTT, 200 mmol DTT and using oligo-dT as 
primers. Increased NGF (FIG. 5) or BDNF (FIG. 11A and FIG. 11B) mRNA was 
observed in pNGF- and pBDNF-transfected cells, respectively as compared to 
sham transfections. To check for possible DNA contamination during RNA 
preparation, RNA samples were included without performing reverse 
transcription; no DNA was detected in these control samples. 
For PCR.TM., one pair of forward and reverse primers of NGF were used. The 
sequence of NGF/5 primer was 5'-GGCATGCTGGACCCAAGCTC-5' (SEQ ID NO:3), and 
of NGF/3, was 5'-GCGCTTGCTCCGGTGAGTCC-3' (SEQ ID NO:4) (Giordano et al., 
1991). Total DNA and 40 pmol of primer were used for PCR.TM.. PCR.TM. was 
carried out in a programmable bearing block (Perkin-Elmer, Norwalk, Conn.) 
using cycles consisting of denaturation at 95.degree. C. for 1 min, 
followed by annealing at 35.degree. C. for 1 min, and DNA extension at 
72.degree. C. for 2 min (Yang et al., 1993). 
After 25 cycles of PCR.TM., samples were electrophoresed on 1.5% agarose 
gels. Gels were stained with ethidium bromide and photographed under UV 
light. NGF mRNA expression was increased in NGF DNA-transfected cultures 
compared to liposome-only treated cultures, and medium-only control 
cultures. The 100-bp DNA ladder from Gibco BRL (Grand Island, N.Y.) was 
used as molecular weight markers. 
Because of interest in injury mechanisms in the hippocampus and the 
preferential vulnerability of the hippocampus to traumatic or ischemic 
brain injury (Jenkins et al., 1989; Lyeth et al., 1990), mixed primary 
septo-hippocampal cell cultures were used for in vitro studies of 
liposome-mediated gene transfection. Cultures were incubated for one week 
prior to transfection. By is that time, astrocytes reached confluence and 
were no longer actively multiplying, while neurons were well 
differentiated and stable. 
2. ELISA Analysis of NGF Protein 
NGF protein levels were examined two days after liposome-mediated NGF 
transfection using an antibody-sandwich ELISA assay. NGF concentrations 
were quantified against a standard concentration curve of pure isolated 
murine NGF. Cells were treated with lysis buffer and extracted on ice for 
10 min. Extractions were centrifuged at 20,000.times.g for 15 min at 
4.degree. C. Wells were coated with 0.25 .mu.g/ml anti-NGF antibodies 
(Boehringer Mannheim Corp., Ind.). A standard NGF dilution series was 
prepared. Diluted homogenate or medium (100 .mu.l) and standard NGF 
dilutions (100 .mu.l) were added to the antibody-coated wells and 
incubated overnight at 4.degree. C. After washing the plate with water and 
blocking buffer, antibody-.beta.-Gal-conjugate solution (100 .mu.l) was 
added to each well and incubated (4 h at 37.degree. C.). After another 
wash with blocking buffer, substrate solution (200 .mu.l) was added to 
each well and incubated (37.degree. C. for 2 h). Plates were read at 570 
nm. A graph of these results showed significant (P&lt;0.01) increases in NGF 
protein in septo-hippocampal cultures 2 days after transfection (values 
represent means=S.E.M.; n=4). These studies detected dramatic increases in 
NGF protein in cell pallets from transfected septo-hippocampal cultures. 
Three days after NGF gene transfection, robust increases of NGF protein 
were detected by ELISA in the cell culture medium. The secreted form of 
NGF in the medium could still be detected two weeks after pNGF 
transfection. Since we routinely exchange the medium 3 time a week after 
gene transfection, the consistent detection of the secreted form of NGF in 
the medium suggests that septo-hippocampal cells express and secrete NGF 
for at least two weeks after liposome-mediated gene transfection. 
ELISA analysis of NGF protein in culture medium: Three days after NGF gene 
transfection, NGF protein was increased ten fold in the medium from NGF 
DNA transfected cultures. Increased secreted NGF could be detected in the 
medium two weeks after NGF DNA transfection (values represent 
means=S.E.M.; n=4). The medium was exchanged 3 times a week after gene 
transfection. 
3. PC12 Confirmed NGF Activity Following Transfection 
Rat pheochromocytoma (PC12) cells were used to confirm the specific 
biological activity of NGF in medium conditioned by cell cultures 
transfected with NGF DNA. PC12 cell medium was removed three hours after 
plating, a sufficient amount of time for cells to attach to wells, and 
replaced with 0.5 ml of conditioned medium collected from cultures three 
days following liposome-mediated NGF DNA transfection. NGF (20 ng/ml) was 
added to sister wells to assay the cells' response to exogenous NGF. 
Thirty-three hours later, cells were observed for the presence of neurite 
outgrowth. The secreted form of NGF in the NGF DNA transfected cell medium 
produced biological effects similar to those of NGF isolated from mouse 
submaxillary gland. However, medium from control cells incubated only with 
liposomes did not produce neurotrophic effects. 
Assay of bioactivity of NGF: representative photomicrographs of changes in 
PC12 cell morphology after 33 h in conditioned media from primary 
septo-hippocampal cultures. Rat PC12 cells were plated on a 24-well plate 
(pre-coated with 50 .mu.g/ml poly-D-lysine in RPM-1640 culture medium at a 
density of 2.times.10.sup.6 cell/0.5 ml). Prominent neuritic outgrowth and 
associated growth cones were produced by the medium from NGF DNA 
transfected cultures. These results were similar to those obtained 
following administration of exogenous NGF. 
These results represent the first reported use of liposome-mediated NGF 
transfection in post-mitotic central nervous system cell cultures. The 
levels of NGF protein expressed in our transfection system are 
particularly high, persist for at least 14 days and elicit prominent 
neurotrophic effects such as neurite growth and growth cone formation. The 
persistent secretion of large amounts of NGF in media after pNGF 
transfection suggests the potential utility of neurotrophin gene 
transfection for treatment of neuronal injury or degenerative disorders. 
EXAMPLE 7 
MIXED PRIMARY SEPTO-HIPPOCAM CELL CULTURES 
Hippocampal and septal neurons are prepared from the brain of 18-day old 
Sprague-Dawley rat fetuses using the method of Banker and Cowan (1977) 
(Banker and Cowan, 1977), but without trypsinization. After washing the 
cells are dissociated by repeated passage through a flame constricted 
Pasteur pipette, collected by centrifugation, resuspended in D-MEM 
supplemented with 10% fetal calf serum and then plated on poly-L-lysine 
coated 24-well plates (1.09.times.10.sup.5 cells/well). Cells for in situ 
hybridization are plated similarly but in 8-well Nunc Lab-tek chamber 
slides. Cultures are kept in a humidified CO.sub.2 incubator at 37.degree. 
C. After 5 days of culture the media is changed into DMEM-based B18 medium 
(Brewer and Cotman, 1989). Subsequent media replacement is carried out 
twice a week. Cultures are allowed at least one week of incubation prior 
to transfection. By that time, astrocytes have reached confluence and are 
no longer actively multiplying, while neurons are well differentiated and 
stable. 
EXAMPLE 8 
IN VIVO TRANSFECTION FOLLOWING TBI USING RODENT MODELS 
Two models of traumatic brain injury have been developed: The fluid 
percussion model of traumatic brain injury injects a small volume of 
saline epidurally into the close cranial cavity of rats (Dixon et al., 
1987). The inventors have employed this device to model moderate diffuse 
brain injury. The second model of brain injury employs pneumatically 
controlled impact to the exposed cortical surface of rats (Dixon et al., 
1991). The inventors employ this device to model severe head injury 
associated with mass lesions and greater frequency of diffuse axonal 
injury than observed with the fluid percussion device. 
Both of these devices have been used to examine the effects of varying 
severities of traumatic brain injury on gene transfection. Both of these 
models were developed in the inventors' laboratories and have been widely 
employed by investigators of central nervous system trauma. 
1. Fluid Percussion Injury 
Animals are surgically prepared for fluid percussion injury under sodium 
pentobarbital anesthesia (54 mg/kg, i.p.) 48 hours prior to trauma (for 
details, see Dixon et al., 1987). Surgical procedures are performed under 
sterile conditions in a laboratory site dedicated to rat surgery. A 4.8-mm 
diameter craniotomy is performed over the sagittal suture midway between 
lambda and bregma. Two stainless-steel skull screws (2-56.times.9.5 mm) 
are placed into burr holes 1 m rostral to bregma and 1 mm caudal to 
lambda. 
A rigid plastic injury tube (modified Leur-loc syringe hub, 2.6 mm inside 
diameter) is placed over the exposed dura and bonded to the skull with 
cyanoacrylate adhesive. Dental acrylic is then poured around the injury 
tube and skull screws and the tube is then sealed with Gelfoam. The scalp 
is sutures closed and Bacitracin ointment is applied over the incision. 
Animals are returned to their home cage (one animal per cage) after they 
have recovered from anesthesia. 
All rats are anesthetized with isoflurane (4%) and a 2:1 N.sub.2 O/O.sub.2 
mixture prior to injury. Animals will be observed for the presence of 
convulsions and apnea. Core body temperature is monitored using a rectal 
probe and maintained at 37.degree.-38.degree. C. The fluid percussion 
device consists of a Plexiglas cylindrical reservoir bounded at one end by 
a Plexiglas piston mounted on O-rings. The entire system is filled with 
isotonic saline. Injury is induced by the decent of a metal pendulum 
striking the piston. This injects a small volume of saline epidurally into 
the closed cranial cavity, producing a brief displacement and deformation 
of neural tissue. The resulting pressure pulse is monitored on a storage 
oscilloscope (Textronix 5111). 
2. Cortical Impact Injury 
The injury device was modified from similar devices developed at the 
Biomedical Science Department of the General Motors Research Laboratories 
in Warren, Mich. The pneumatic impactor consists of a small (1.975 cm) 
bore, double-acting, stroke-constrained, pneumatic cylinder with a 5.0 cm 
stroke (for details, see Dixon et al., 1991). The cylinder is rigidly 
mounted on a crossbar. The lower rod end has an impactor tip attached. The 
upper rod end is attached to the transducer core of a linear velocity 
differential transformer (LVDT) (Shaevitz Model 500 HR). The impact 
velocity can be adjusted by controlled gas pressure. Impact velocity is 
directly measured by the LVDT which produces an analog signal that is 
recorded for analysis of time/displacement parameters of the impact. 
All animals are initially anesthetized with 4% isoflurane and a 2:1 N.sub.2 
O/O.sub.2 mixture. Following endotracheal intubation, rats are 
mechanically ventilated with a 2% isoflurane mixture. The rats are mounted 
in the injury device's stereotaxic frame. A midline incision is made, soft 
tissues reflected, and a craniectomy performed. Core body temperature is 
monitored continuously by a rectal thermistor probe and maintained at 
37-38.degree. C. 
EXAMPLE 9 
DETERMINATION OF TIME COURSE OF TRANSFECTION 
To determine the time course of transfection, in situ hybridization and 
immunohistochemical studies of the dorsal hippocampus is performed 
following control injections of liposomes alone and at one day, three 
days, one week, one month, two months and three months following 
injections of optimal ratios of cDNA to liposomes for NGF and BDNF. Based 
on regional analyses, RT-PCR and ELISA studies are conducted at selected 
time points. At these same selected time points, immunohistochemical 
analyses of neurotrophin receptors (p75.sup.NGFR, p140.sup.trkA, 
p145.sup.trkB) and assay bioactivity of NGF and BDNF are also performed. 
The inventors' choice of time points was based on the observation that 
liposome mediated gene transfection is transient and has been reported to 
last up to two months. 
EXAMPLE 10 
SHORT-TERM POTASSIUM DEPOLARIZATION TRANSIENTLY DECREASES NEUROFILAMENT 
PROTEINS IN MIXED SEPTO-HIPPOCAM CULTURES 
Chronic (3-6 hour) potassium depolarization of cultured neurons enhances 
maturation of the neuronal cytoskeleton. However, traumatic brain injury, 
which is accompanied by transient (.about.3 min.) potassium depolarization 
produces significant cytoskeletal degradation including loss of axonal NF 
protein. To investigate the effect of short-term depolarization on 
neurofilament proteins and determine calcium's role in that effect, mixed 
septo-hippocampal cultures were exposed for 6 min. to 60 mM KCl in the 
presence of Ca.sup.++ concentrations from 1.8 to 5.8 mM. Cultures were 
depolarized at 10 days in vitro when neuronal arborization was extensive 
and astrocytes were confluent. At 9 to 10 days after depolarization, 
cultures were lysed and homogenates were analyzed via SDS-PAGE Western 
blotting for alterations in NF150 and NF68. 2-D densitometry was used for 
quantitation. 
Neurofilament proteins were decreased in all treated cultures. Initial 
results indicate levels returned to normal by 10 days after insult 
following treatment with up to 2.8 mM Ca.sup.++. This decrease was 
Ca.sup.++ -dependent and exhibited different time courses for the two 
proteins examined. Thus, short-term depolarization can temporarily 
decrease levels of neurofilament proteins. Ca.sup.++ -dependent disruption 
of the axonal cytoskeleton may play a role in the reversible functional 
deficits observed in rat models of traumatic brain injury. 
EXAMPLE 11 
NEUROFILAMENT 68 AND NEUROFILAMENT 200 PROTEIN LEVELS DECREASE AFTER 
TRAUMATIC BRAIN INJURY 
A. MATERIALS AND METHODS 
1. Cortical Impact and Tissue Dissection Nine groups (n=6-8 per group) of 
male Sprague-Dawley rats (250-350 g) were used in this study. Groups 
included naive animals and the following post-injury groups: sham 3 h, 
injured 3 h, sham 1 day, injured 1 day, sham 7 days, injured 7 days, sham 
14 days, and injured 14 days. For induction of severe TBI, a controlled 
cortical impact device described previously (Dixon et al., 1991) was 
utilized. Briefly, rats were intubated and anesthetized with 2% halothane 
in a 2:1 mixture of N.sub.2 O/O.sub.2. Two 7 mm diameter craniotomies were 
performed adjacent to the central suture, midway between lambda and 
bregma. The dura was kept intact. Injury was induced by impacting the 
right exposed cortex with a 6 mm diameter tip at a rate of 6 m/sec and a 2 
mm compression (Dixon et al., 1991). Sham-injured animals underwent 
identical surgical procedures but did not receive impact injury. Following 
cortical impact, animals were extubated and immediately assessed for 
recovery of reflexes (Dixon et al., 1991). Animals whose righting response 
did not exceed 5 min were omitted from the study. 
Following assessment and at the appropriate time interval, animals were 
killed under halothane anesthesia and immediately prepared for tissue 
microdissection. Sections of the hippocampus and cortex directly beneath 
and craniotomies were removed. Cortex was selected for study because, 
ipsilaterally, it is the locus of injury, and contralaterally, it may 
represent a contre coup damage site resulting from tissue extrusion at 
impact (Meaney et al., 1992). The underlying hippocampal tissue was 
examined because of its established preferential vulnerability after TBI 
(Hayes et al., 1992; Taft et al., 1992). The dorsal hippocampus was 
microdissected bilaterally. Excision of the parietal cortices beneath the 
craniotomies extended approximately 4 mm laterally, approximately 7 mm 
rostrocaudally, and to a depth extending to the white matter. All tissue 
was frozen immediately in liquid N.sub.2. 
2. Sample Processing and Gel Electrophoresis 
The microdissected tissue homogenized at 4.degree. C. in an ice-cold 
homogenization buffer containing 20 mM PIPES (pH 7.1), 2 mM EGTA, 1 mM 
EDTA, 1 mM dithiothreitol, 0.3 mM phenylmethylsulfonylfluoride (PMSF), and 
0.1 mM leupeptin. The presence of chelators and protease inhibitors 
prevents endogenous in vitro activation of proteases and subsequent 
artifactual degradation of the NF proteins in vitro. Proteins were 
assessed for concentration by the Bradford procedure (Bradford, 1976) and 
balanced for protein content. Samples were then solubilized by 2;1 
dilution in sodium dodecyl sulfate (SDS)-containing stop solution (600 mM 
Tris, 200 mM EDTA, 10% sucrose, 3% SDS) and heated for 5 min at 85.degree. 
C. Proteins were reduced with .beta.-mercaptoethanol and resolved by SDS 
polyacrylamide gel electrophoresis (PAGE). A vertical electrophoresis 
chamber using a 4.5% acrylamide stacking gel over 6% acrylamide resolving 
gel was routinely employed. Typically, 50-75 .mu.g of sample protein were 
run in each lane at constant current (120 mA) for 2.5 h. All sample lanes 
on each gel contained identical amounts of protein. To insure consistency 
of gel loading, silver staining of identical sister gels was performed and 
Ponceau red staining of each blot was conducted prior to immunolabeling. 
To minimize gel-to-gel comparisons, time course gels were routinely 
performed to observe NF changes post-injury with all sampling times 
contained on a single gel. 
3. Quantitative Immunoreactivity of NF68 and NF200 
Immediately after separation of the proteins by SDS-PAGE, proteins were 
transferred to nitrocellulose membrane by Western blotting technique 
(Towbin et al., 1979). Lateral transfer was conducted between two plate 
electrodes and a transfer buffer containing 62.5 mM glycine, 50 mM Tris, 
and 10% methanol was utilized. Transfer occurred at a constant current of 
approximately 365 mA at 4.degree. C. Incubation in primary antibody was 
performed with either anti-NF68 (Sigma N5139) or anti-NF200 (Sigma N0142) 
monoclonal antibodies. 
NF68 and NF200 were chosen for analysis because they are, respectively, the 
most important structural and cross-linking elements of neurofilaments. 
Both monoclonals recognize phosphorylated and non-phosphorylated 
neurofilament epitopes. Visualization was performed with an 
avidin-alkaline phosphate kit using 5-bromo-4-chloro-3-indolyl 
phosphate/nitroblue tetrazolium (BCIP/NBT) as the active chromogen. Using 
this procedure, the anti-NF68 monoclonal labels a single band having an 
apparent M.sub.r of 68 kDa, and the NF200 monoclonal produces a somewhat 
smeared band having an apparent M.sub.r of .gtoreq.200 kDa. The smearing 
of NF200 is caused by the physiochemical properties of this long, rigid 
protein and its extensive, variable level of phosphorylation (Julien and 
Mushynski, 1982; Nixon and Sihang, 1991). 
4. Statistical Analysis 
The studies described were performed with 6-8 rats. Each sample was 
performed in duplicate for a total of 12-16 data points per condition. 
Quantitative analysis of the proteins employed a computer-assisted, linear 
scanning densitometer (GS300, Hoefer Scientific). All quantitative 
readings were performed within a linear range of optical densities. All 
lanes on immunoblots were scanned in the dark under optimal conditions 
three times, and the results were averaged for inclusion in data analysis. 
Lanes that contained prominent gel artifacts were excluded from further 
data analysis. 
Statistical significance of NF immunoreactivities was evaluated using an 
independent t-test analysis of the pooled data and a post-hoc Tukey-Honest 
significant difference (HSD) analysis of variance (ANOVA) to detect 
percentage differences by group analysis for all gels analyzed. All values 
were expressed as percent change relative to naive values on each 
immunoblot. Data were considered significant if p&lt;0.05. 
B. RESULTS 
1. Sham Injury Does Not Affect NF68 and NF200 Levels 
NF68 and NF200 protein levels from sham-injured animals at all time points 
were compared with levels from naive rats to determine the effect of 
animal surgery (craniotomies) in the absence of TBI. Both hippocampal and 
cortical tissues were examined, ipsilaterally and contralaterally. NF68 
and NF200 levels from naive cortex did not differ significantly from 
cortices of sham-injured animals at any post-injury time investigated 
(p&lt;0.05). Similarly, NF68 and NF200 also were unchanged in sham-injured 
hippocampal tissue when compared with naive hippocampi (p&lt;0.05) (Table 2). 
Hence, sham injury had no significant influence on NF68 or NF200 protein 
levels in either the cortex or the hippocampus at any time point 
investigated. 
The effect of lateral cortical impact injury on the levels of axonal 
cytoskeletal proteins was examined in adult rats. TBl caused a significant 
decrease in the protein levels of two major neurofilament (NF) proteins, 
NF68 and NF200. By quantitating immunoreactivity measurements of Western, 
blots NF68 and NF200 levels were determined in homogenates of hippocampal 
and cortical tissue taken at several intervals post-injury. Sham injury 
had no effect on NF protein levels. However, injury was associated with a 
significant (up to 41.8%) loss of NF68, restricted to the cortex 
ipsilateral to the injury site. NF68 loss was detectable as early as three 
hours and as late as two weeks post-injury. 
Similarly, TBl induced a decrease in NF200 protein, although losses were 
observed both ipsilateral (71.7%) and contralateral (66.9%) to the injury 
site. No loss of NF68 or NF200 protein was detected in hippocampal samples 
obtained from the same injured animals. An increase in the presence of 
lower molecular weight NF68 immunopositive bands was associated with the 
decrease of NF68 in the injured ipsilateral cortex. This NF68 antigenicity 
pattern suggests the production of NF68 breakdown products (BDPs) caused 
by the pathological activation of neuronal proteases such as calpain. 
Putative NF68 BDPs increased significantly until one day post-injury, 
suggesting that NF degradation may be ongoing and indicating that a 
potential therapeutic window may exist within the first 24 hrs 
post-injury. In summary, these data identify specific biochemical 
alterations of the axonal cytoskeleton following TBl. 
EXAMPLE 12 
EXTRACELLULAR CHOLINE LEVELS AND [.sup.3 H]-CHOLINE UPTAKE IN HIPPOCAMPUS 
2-WEEKS AFTER CORTICAL IMT INJURY IN RATS 
Chronic spatial memory deficits following experimental traumatic brain 
injury may be, in part, attributable to deficits in central cholinergic 
neurotransmission. These studies examined the rate-limiting factor in 
acetylcholine synthesis; availability and neuronal uptake of its 
precursor, choline. In the first study microdialysis was used to measure 
extracellular choline levels within the dorsal hippocampus at 2-weeks 
post-injury, an interval associated with spatial memory deficits. Ten rats 
were injured by lateral cortical impact. Ten additional rats served as 
sham controls. Under anesthesia, a microdialysis probe (3 mm tip) was 
placed into their dorsal hippocampus and perfused with artificial cerebro 
spinal fluid CSF. Samples were collected for 20 minutes (min) and measured 
by HPLC. The data showed no difference in basal choline levels between 
injured (3.86.+-.0.53 pmol/20 min) and sham (3.35.+-.0.50) rats. 
In a second study high-affinity [.sup.3 H]-choline uptake was measured in a 
synaptosomal preparation of hippocampal tissue removed from 11 injured and 
12 sham rats 2-weeks following TBl. Significant differences were found in 
the maximum velocity of choline uptake (Vmax) between injured (46.2.+-.2.3 
pmol/mg/5 min) and sham (58.8.+-.2.1) rats, while no differences in 
affinity constants (K.sub.m) were found. The results suggest that 
post-traumatic cholinergic deficits are not attributable to decreased 
availability of choline, but may be associated with either a decreased 
ability of cholinergic neurons to take up choline, or a loss of 
cholinergic neurons. 
EXAMPLE 13 
VISUALIZATION OF ACUTE STRUCTURAL DERANGEMENTS OF CORTICAL NEURONS IN THE 
RAT FOLLOWING TBI USING NF IMMUNOFLUORESCENCE 
Quantitative Western blot studies demonstrated significant losses of 
neurofilament 68 (NF68) and neurofilament 200 (NF200), as well as the 
presence of low molecular weight proteolytic fragments as early as 3 hours 
and for as long as two weeks following lateral cortical impact injury in 
rats. Three hours following injury, NF68 decreased 23% and NF200 decreased 
50% in the ipsilateral cortex. In the contralateral cortex, TBI produced 
significant loss (65%) of only NF200 3 hours after injury. Consequently, 
NF immunofluorescence was used to study the morphological correlates of 
early NF protein decreases observed 3 hrs post TBI. 
Using anti-NF200 (Sigma N52) and anti-NF68 (Sigma NR4) antibodies that 
recognize their appropriate subunits independent of phosphorylation state, 
total NF protein was detected regardless of post-translational 
modifications. Secondary anti-mouse IgG was conjugated with either FITC or 
Rhodamine. TBI induced differential alterations in NF68 and NF200 
immunolabeling which were restricted to the ipsilateral contusion site 
(2-4 mm lateral from the middle longitudinal tissue [MLF[) as well as a 
focal contralateral contusion site (1-2 mm from MLF). 
NF200 immunofluorescence revealed a prominent fragmented appearance of the 
apical dendrites within pyramidal neuronal layers 3 and 5, as well as a 
loss of fine dendritic arborization within layer 1. NF68 
immunofluorescence detected subtle and less severe ultrastructural changes 
such as focal vacuolization along apical dendrites, although 
macrostructural changes (i.e., broken apical dendrites) also occurred to a 
lesser degree. Although axonal alternations were observed with anti-NF68 
and anti-NF200 in the corpus callosum and other white matter areas, axonal 
alterations were markedly less than the acute NF immunofluorescence 
changes seen in dendritic regions. 
Collectively, these observations suggest that early NF protein loss is 
observed in dendrites, and to a lesser extent, in axons following injury. 
Since neurofilaments are structural proteins found abundantly in dendritic 
as well as axonal processes, diffuse injury to neuronal processes may be 
an important morphological feature of TBI. Immunohistochemical studies 
were employed to exam the temporal and regional characteristics of this 
diffuse process injury (DPI). 
EXAMPLE 14 
AMYLOID PROTEIN PRECURSOR LEVELS ARE ALTERED IN RAT HIPPOCAMPUS AND CORTEX 
FOLLOWING TRAUMATIC BRAIN INJURY 
Increases in the diffuse deposition of AS protein can occur following 
traumatic brain injury in humans. Furthermore, levels of the precursor to 
AS protein (amyloid protein precursor: APP) are increased in rodent brain 
following CNS lesion. In order to investigate the effect of traumatic 
brain injury on APP levels, homogenates of rat cortex and hippocampus were 
prepared 3 h, 1 day, 7 days, or 14 days after lateral cortical impact 
injury. SDS-PAGE Western blots were immunostained using an antibody which 
recognizes all three main forms of the amyloid protein precursor (22C11, 
Boehringer-Mannheim, Indianapolis, Ind.). 2-D densitometric scanning was 
used to quantify changes in protein levels. 
Data indicated that APP levels were significantly decreased at 2 weeks in 
cortex ipsilateral to injury. In contrast, a significant increase was 
observed in ipsilateral hippocampus 1 day after injury. These results 
indicate that alterations in APP after traumatic brain injury are 
regionally specific. Thus, mechanisms mediating TBl-induced changes in APP 
levels may differ in various regions of the rodent brain. 
EXAMPLE 15 
EFFECTS OF LATERAL CORTICAL IMT INJURY ON AXONAL PROTEINS 
The effect of lateral cortical impact injury on the levels of axonal 
cytoskeletal proteins has been examined in adult rats. Traumatic brain 
injury (TBI) causes a significant decrease in the protein levels of two 
prominent neurofilament (NF) proteins, NF68 and NF200. Quantiative 
immunoreactivity measurements were performed using Western blots to 
examine NF68 and NF200 levels in homogenates of hippocampal and cortical 
tissue taken at several intervals post-injury. Sham injury had no effect 
on NF protein levels. However, injury was associated with a significant 
loss of NF68, restricted to the cortex ipsilateral to the injury site. 
1. Injury Results in Significant loss of NF68 and NF200 
NF68 loss was detectable as early as 3 h and lasted at least 2 weeks 
post-injury. Similarly, TBI induced a decrease in NF200 protein, although 
losses were observed both ipsilateral and contralateral to the injury 
site. No loss of NF68 or NF200 protein was detected in hippocampal samples 
obtained from the same injured animals. An increase in the presence of 
lower molecular weight (MW) NF68 immunopositive bands was associated with 
the decrease of NF68 in the ipsilateral cortex. This NF68 antigenicity 
pattern suggests the production of NF68 breakdown products caused by the 
pathologic activation of neuronal proteases, such as calpain. Putative 
NF68 breakdown products increase significantly until 1 day post-injury, 
suggesting that NF degradation may be ongoing until that time and 
indicating that a potential therapeutic window may exist within the first 
24 h post-injury. 
2. TBI Decreases NF68 Levels Only in Ipsilateral Cortex 
The effect of lateral cortical impact injury on NF68 immunoreactivity in 
cortical tissue was examined at several intervals post-injury. Naive, 
sham-injured, and cortical impact-injured samples were examined for NF68 
protein content by SDS-PAGE and quantitative Western immunoreactivity at 3 
h, 1 day, 7 days, and 14 days after injury. Proteins were visualized on 
nitrocellulose transfers using an anti-NF68 monoclonal antibody. A 
significant decrease in NF68 protein was noted at all postinjury time 
points examined. The decrease in NF68 antigenicity was accompanied by the 
appearance of two lower MW immunopositive bands at 56 and 52 kDa. 
In the contralateral cortex, no significant change in NF68 levels was noted 
between sham and injured samples. The most prominent loss (58.2% of sham 
controls) occurred at 7 days post-injury. The process of NF68 loss was 
induced within 3 h post-injury, but continued reduction of NF68 occurred 
beyond 1 week. In addition, NF68 levels at 1 day (63.9% of sham controls) 
were significantly lower than the 3 h value (78.9% of sham controls) 
(p&lt;0.04). Post-hoc Tukey-HSD ANOVA analysis (% difference group) for all 
gels at p&lt;0.05 also showed significance at all time points except 3 h. In 
addition to labeling of the prominent 68 kDa band, additional 
immunopositive bands were found in samples from injured cortex. These 
lower MW immunopositive bands appeared prominently at 56 kDa and 52 kDa 
and are accompanied by other minor staining bands in the same MW range 
(FIG. 9A and FIG. 9B) 
TABLE 2 
______________________________________ 
NF68 AND NF200 LEVELS IN HIPPOCAM SAMPLES.sup.a 
3 h 1 Day 7 Days 14 Days 
______________________________________ 
NF68 Protein Levels 
Ipsilateral 
Sham 99.9 .+-. 6.6 
94.6 .+-. 1.6 
91.8 .+-. 3.3 
93.1 .+-. 4.5 
Injured 88.9 .+-. 5.9 
86.8 .+-. 4.8 
85.7 .+-. 2.0 
84.5 .+-. 3.0 
Contralateral 
Sham 101.9 .+-. 5.3 
97.2 .+-. 4.1 
95.1 .+-. 7.6 
94.4 .+-. 3.6 
Injured 93.05 .+-. 1.9 
92.0 .+-. 6.4 
95.9 .+-. 4.3 
90.3 .+-. 3.7 
NF200 Protein Levels 
Ipsilateral 
Sham 94.1 .+-. 6.1 
96.6 .+-. 18.0 
103.1 .+-. 15.2 
87.8 .+-. 11.5 
Injured 83.9 .+-. 10.4 
110.9 .+-. 5.5 
87.8 .+-. 11.5 
87.2 .+-. 7.8 
Contralateral 
Sham 94.2 .+-. 8.3 
96.4 .+-. 1.6 
96.9 .+-. 1.6 
78.9 .+-. 9.4 
Injured 105.4 .+-. 6.4 
95.6 .+-. 10.3 
87.7 .+-. 11.6 
86.6 .+-. 4.2 
______________________________________ 
.sup.a NF68 and NF200 levels in hippocampus after severe cortical impact 
TBI. The effect of lateral cortical impact injury on NF68 and NF200 
immunoreactivity in hippocampal tissue was examined at several intervals 
postinjury. Naive, shaminjured, and cortical impactinjured samples were 
examined for neurofilament protein content by SDSPAGE and quantitative 
Western immunoreactivity at 3 h, 1 day, 7 days, and 14 days after injury. 
Proteins were visualized on nitrocellulose transfers #using appropriate 
monoclonal antibodies. Immunoblots were analyzed quantitatively by 
computerassisted scanning densitometry. Data are expressed as percentage 
relative to naive controls. No significant changes in NF68 or NF200 
protein levels are detected in hippocampal tissue either ipsilateral or 
contralateral to the injury site. Statistical analysis of the data 
included an independent ttest of the pooled data and a post hoc TukeyHSD 
ANOVA #to detect percentage difference by group analysis for all gels 
analyzed. All values were p &lt; .05. Thus, no injured groups showed 
significant differences from the shaminjured controls. 
Cortical impact injury was associated with the onset of a pronounced and 
prolonged loss of NF68 in the ipsilateral cortex. NF68 levels in the 
ipsilateral cortex were significantly reduced at 3 h, 1 day, 7 days, and 
14 days. NF68 levels in the contralateral cortex did not differ from sham 
controls at any time point studied. No prominent lower MW immunopositive 
bands were seen in the contralateral cortical samples. Thus, the data 
suggest a clear differential reduction in NF68 protein selective to the 
ipsilateral cortex caused by TBI. 
3. TBI Decreases NF200 in Cortical Tissue 
The effect of lateral cortical impact injury on NF200 immunoreactivity in 
cortical tissue was also examined at several intervals post-injury. Naive, 
sham-injured, and cortical impact-injured samples were examined for NF200 
protein content as described at 3 h, 1 day, 7 days, and 14 days after 
injury. Proteins were visualized on nitro-cellulose transfers using an 
anti-NF200 monoclonal antibody. In the ipsilateral cortex, a significant 
decrease in NF200 protein was noted at all post-injury time points 
examined. In the contralateral cortex, a reduction of NF200 levels was 
noted between sham and injured samples at all intervals post-injury. 
TBI also caused a dramatic reduction in cortical NF200 protein levels. 
NF200 loss was most prominent in the ipsilateral cortex, but unlike the 
NF68 changes, significant loss of NF20 occurred in the contralateral 
cortex as well. In the ipsilateral cortex, NF200 loss was seen at 3 h and 
remained significantly reduced as long as 2 weeks post-injury. The 
greatest loss (28.3% of sham control) was observed at 7 days post-injury. 
NF200 immunoreactivity in the contralateral cortex also showed a consistent 
TBI-induced reduction. Although each time point analyzed showed reduction 
of NF200 levels relative to sham controls, significant decreases occurred 
only at 3 h and 14 days. The increased variability of NF200 sampling 
suggests a more diverse insult to NF protein contralaterally. 
4. TBI Does Not Produce Derangements of NF Proteins in Hippocampal Tissues 
NF68 and NF200 immunoreactivity was performed on the corresponding 
underlying hippocampal tissue for all animals studied. NF68 
immunoreactivity in the ipsilateral and contralateral hemispheres did not 
show any significant differences, (p&lt;0.05) compared with sham-injured 
controls. Similarly, NF200 demonstrated no significant loss in either 
hemisphere of injured animals compared with sham-injured animals (Table 
2). The low MW NF68 immunopositive bands were not seen in hippocampal 
samples. 
5. Post-TBI NF Protein Loss Is Associated with the Presence of NF Breakdown 
Products 
Immunoanalysis of NF68 on Western blots demonstrated a prominent single 
band at 68 kDa in naive, sham, and injured animals. In injured samples, 
however, the decrease in NF68 levels was associated with the appearance of 
lower MW immunopositive bands (FIG. 9A and FIG. 9B). The minor bands were 
found at molecular weights of 56 kDa and 52 kDa. The presence of the lower 
MW bands associated with TBI and the concomitant reduction of NF68 suggest 
the appearance of lower MW breakdown products (BDPs) caused by the 
post-injury activation of proteases. The low MW immunopositive proteins 
appeared at 3 h post-injury and attained their highest levels at 1 day 
(FIG. 9B). These bands were markedly reduced in 7 and 14 days samples. The 
presence of low MW immunopositive bands has been reported previously 
following both in vitro and in vivo proteolysis of NF68 (Schlaepfer et 
al., 1984; Schalepfer and Zimmerman, 1985). 
6. The Role of Cytoskeletal Proteins in Trauma 
Studies have addressed the relationships between changes in axonal and 
dendritic cytoskeletal proteins and the morphopathologic outcome of 
trauma. These quantitative analyses indicate that NF changes precede 
neuronal cell death and, further, may not be exclusively associated with 
cell death. For example, cortical NF68 levels were significantly decreased 
at 3 h post-injury, well in advance of cellular necrosis in the cortex. In 
addition, cortical NF200 loss occurred contralateral to the impact site, 
in the absence of substantial cell death. 
Similarly, loss of cortical NF200 has been reported contralaterally to 
middle cerebral artery occlusion in rats where no cell death occurred but 
which may contribute to postischemic symptomatology (Inuzuka et al., 
1990b). Subsequent studies directly linked to neuropathology may determine 
whether the NF losses reported are initial components of axonal damage and 
separation or are the beginnings of Wallerian degeneration of the distal 
axonal segment of the already severed axon. Alternatively, decreases in NF 
may represent persistent sublethal alteration in the cytoskeleton not 
associated with classic morphopathologic features of traumatic axonal 
injury. 
Even in the absence of cell death or axonal disruption, the NF changes 
identified may have important consequences that compromise neuronal 
function and may contribute to sublethal neuronal injury. NF68 and NF200 
are key elements of neurofilaments, and extensive loss of these proteins 
after TBI may compromise neurofilament function in injured neurons. Such 
insults may be critical to neuronal viability and normal function because 
neurofilaments appear to play a crucial role in the maintenance of axonal 
and dendritic structure, caliber, axoplasmic transport, and other 
functions (Hoffman and Lasek, 1975; Mellgren and Murachi, 1990; Oliver et 
al., 1989). These actions may be significantly impaired post-injury with 
the losses of NF protein reported here. 
NF68 loss at 1 day post-injury is significantly greater than the loss 
observed at 3 h post-injury. This difference could indicate that the 
pathologic processes causing NF loss are incomplete 3 h post-injury and 
are ongoing at that time. Because the NF loss at 1, 7, and 14 days is not 
statistically different, the pathologic causes of NF loss may have peaked 
at these times. These observations are consistent with the appearance of 
NF68 BDPs, which also peak 1 day post-injury. In experimental models of 
ischemia, administration of anti-calpain compounds has proven effective in 
preventing ischemia-induced cytoskeletal protein loss (Inuzuka et al., 
1990a; Lee et al., 1991; Siman et al., 1989). In models of TBI, treatment 
with moderate hypothermia reduces injury-associated MAP2 loss (Taft et 
al., 1993). Thus, the changes in NF protein levels may be amenable to 
therapeutic intervention in the first 24 h post-TBI. The time course of NF 
changes further suggest that NF loss will not recover to normal (sham 
control) levels within the first 2 weeks following insult. The examination 
of longer post-injury intervals may be necessary to determine whether 
neurons can independently recover NF protein levels following severe 
cortical impact TBI. 
7. Conclusions 
Several important conclusions were derived from these studies. First, the 
pattern and temporal progression of NF68 and NF200 loss were similar. 
Reductions in NF levels were observed within 3 h post-injury and appear 
unresolved 2 weeks later. Second, the extent of NF protein loss was 
greater in the hemisphere ipsilateral to the injury site compared with the 
contralateral side. For example, a pronounced loss of NF68 was seen 
ipsilaterally, but no loss was observed contralaterally. Third, the loss 
of NF200 was more extensive compared with loss of NF68. In the ipsilateral 
cortex, NF200 loss reached 70% (30% of sham control values), whereas NF68 
loss was 40% (60% of sham control values). Fourth, a higher degree of 
sample-to-sample variability was observed with NF200 compared with NF68. A 
potential cause for the greater loss and higher variability of NF200 
post-TBI may be NF200 subunit disassembly from the core filament, thus 
increasing its availability for phosphorylation or proteolysis or both. 
Further, because NF phosphorylation state influences NF proteolysis by 
calpain (Pant, 1988), variable levels of NF200 phosphorylation from animal 
to animal may contribute significantly to the variability of NF200 
antigenicity. 
EXAMPLE 16 
RESCUE OF NEUROFILAMENT LOSS FROM NEURONAL INJURY BY BDNF GENE TRANSFECTION 
IN PRIMARY SEPTO-HIPPOCAM CELL CULTURE 
Cortical impact injury in rats can produce significant loss of 
neurofilament proteins including medium, low and high molecular weight 
neurofilament proteins (NF-L, NF-M, NF-H). In addition, brief 
depolarization of primary septo-hippocampal cell cultures can also produce 
significant losses of neurofilament proteins. Recent studies indicate that 
BDNF increases neurofilaments in hippocampal cell cultures (Yip et al., 
1993) and increases survival of cortical neurons (Ghosh et al., 1994). 
Using a liposomal mediated system for transfecting cDNA of neurotrophins 
into central nervous system cells primary septo-hippocampal cell cultures 
were transfected to determine the therapeutic potential of BDNF gene 
transfection in facilitating the recovery of neurofilament loss caused by 
depolarization injury. 
Employing a pUC19 based plasmid, rat BDNF cDNA was subcloned into a unique 
NotI site under the control of the CMV promoter. DNA for BDNF was 
complexed with liposomes and transfected into primary septo-hippocampal 
cell cultures one day after depolarization injury (6.0 min depolarization 
with 60 mM KCl and the presence of 2.8 mM Ca.sup.++) (FIG. 11A and FIG. 
11B). Three days after depolarization injury, Western blot and 
immunohistochemical analyses detected significant loss (42% of NF-M and 
NF-H proteins (Sternberger SMI 31 antibody) in untreated cultures (FIG. 
13A and FIG. 13B). However, densitometric scanning of Western blot data 
indicated that BDNF transfection produced a two-fold increase in NF-M/NF-H 
three days following injury as compared to untreated cultures. 
Immunohistochemical studies also detected enhanced NF-M/NF-H 
immunolabeling in injured neurons following BDNF transfection as compared 
to untransfected, injured controls. Thus, BDNF gene transfection may be 
useful as a therapeutic tool for blunting neurofilament loss associated 
with injury to central nervous system neurons. 
EXAMPLE 17 
BDNF TRANSFECTION RESCUES NF IN INJURED ANIMALS 
To demonstrate the effects of TBI on levels of neurofilament protein in 
vivo, NF-H levels were determined in animals following TBI using SDS-PAGE 
and Western analysis. FIG. 13A shows the decrease in NF-H levels which 
occur as a result of TBI. Tissue was dissected from the parietal cortex 
ipsilateral to the site of cortical impact. Two days following cortical 
impact injury, a substantial loss of NF-H protein was detected in the 
injured (I) rat compared to the non-injured (N) control. Densitometric 
scans of the two samples indicated a 38% decrease in NF-H levels following 
cortical impact injury. (In arbitrary units, 4083 density units were 
recorded for the non-injured NF level, and 2614 density units were 
recorded for the injured NF level). 
To demonstrate the successful rescue of NF-H levels in vivo after the 
liposome-cDNA transfection methods of the present invention, neurofilament 
recovery was determined by following the immunoreactivity of NF-H using 
SDS-PAGE and Western analysis. Tissue was dissected from the parietal 
cortex ipsilateral to the site of cortical impact as in FIG. 13A. Compared 
to the control animal (C) which received liposomes alone, the animal 
transfected with the liposome-BDNF cDNA complex (1:3) ratio) (B) showed a 
significant restoration of NF-H protein two days after injury. In both 
cases, injections were made into the ventricle ipsilateral to the injury 
site immediately after injury. An 8% increase in NF-H was detected 2-days 
post-trauma in the BDNF-transfected animal. (By densitometric scans, 
arbitrary units of 5516 and 5862 density units were recorded for NF-H 
level in the injured control, and the injured BDNF-transfected animal, 
respectively. 
EXAMPLE 18 
GENERAL METHODS 
1. .beta.-Galactosidase Activity 
.beta.-Gal activity is visualized in vivo by histochemical staining using 
5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (X-Gal). Culture 
wells are washed with phosphate-buffered saline (PBS) and fixed with 0.5 
ml containing 2% (v/v) paraformaldehyde and 0.2% glutaraldehyde in H.sub.2 
O for 5 min at 4.degree. C. Cells are rinsed again with PBS and then 
stained with 0.5 ml per well of the following solution: 7 mM Na.sub.2 
HPO.sub.4, 23 mM NaH.sub.2 PO.sub.4, 1.3 mM MgCl.sub.2, 3 mM K.sub.3 
Fe(CN).sub.6, 3mM K.sub.4 Fe(CN).sub.6 and 1 mg/ml X-Gal (diluted from a 
40 mg/ml stock solution in dimethylformamide). Cells expressing .beta.-Gal 
are stained blue after incubation at room temperature overnight. 
2. mRNA Assessments 
Cells grown in culture are lysed by adding 0.2 ml of RNAzol B (Cinna/Biotec 
Laboratories, Inc.) per 10.sup.6 cells. The RNA is solubilized by passing 
the lysate several times through a pipette. After adding 0.2 ml chloroform 
per 2 ml, the homogenate is incubated at 4.degree. C. for 15 min, and the 
aqueous phase containing the RNA is transferred to a fresh tube. The 
supernatant is removed after centrifugation (12,000.times.g) for 15 min at 
4.degree. C. RNA pellets are washed with 75% ethanol, dried briefly and 
re-suspended in RNase-free water. 
For in vivo studies, hippocampal tissue around injection sites and/or other 
loci is dissected at 4.degree. C. and homogenized with RNAzol B (2 ml/100 
mg) by a motorized Teflon.TM. pestle homogenizer (10 up-and-down stokes). 
Methods are then similar to those employed for in vitro studies. 
3. Reverse Transcription-Polymerase Chain Reaction (RT-PCR.TM.) 
Reverse transcription is performed using oligo (dt) as primers and M-MLV 
reverse transcriptase (Perkin-Elmer, Norwalk, Conn.). Twenty .mu.g of 
total RNA from each sample is used for cDNA synthesis. The reaction is 
carried out at 42.degree. C. for 2 hours using 20 units of recombinant 
reverse transcriptase devoid of phenol, then with chloroform, followed by 
precipitation with ethanol. The cDNA is dissolved in 50 .mu.l of TE 
buffer. 
For PCR.TM., two pairs of forward and reverse primers for BDNF and NGF are 
used. The sequence of the BDNF/5 primer is 5'-GCAAACATGTCTATGAGGGT-3' (SEQ 
ID NO:1) and BDNF/3 is 5'-GGTCAGTGTACATACACAGG-3' (SEQ ID NO:2); the 
sequence NGF/5 primer is 5'-GGCATGCTGGACCCAAGCTC-3' (SEQ ID NO:3) and 
NGF/3 is 5'-GCGCTTGCTCCGGTGAGTCC-3' (SEQ ID NO:4) (Giordano et al., 1992). 
2 .mu.l cDNA and 40 pmol of primer are used for PCR.TM.. PCR.TM. is 
carried out in a programmable heating block (Perkin-Elmer) using cycles 
consisting of denaturation at 95.degree. C. for 1 min, followed by 
annealing at 55.degree. C. for 1 min and cDNA extension at 72.degree. for 
2 min. 
After amplification, the samples electrophoresed on 1.5% agarose gel. The 
gels are stained with ethidium bromide and photographed under UV light. 
The intensity of PCR.TM. product bands are quantitated by a 
computer-assisted, linear-scanning densitometer in reflectance mode 
(Hoefer Scientific Instruments, San Francisco, Calif.). 
Since previous studies indicated that .beta.-actin mRNA did not change in 
the inventors' injury model (Yang et al., 1993), .beta.-actin mRNA is 
utilized in the same RNA preparation as one internal control for RT-PCR. 
The sequence for the forward primer is 5'-CCTTCCTGGGCATGGAGTCCTG-3' (SEQ 
ID NO:5). The sequence of the reverse primer is 5-GGAGCAATGATCTTGATCTTC-3' 
(SEQ ID NO:6). To check for the possibility of DNA contamination during 
RNA preparation, the same RNA samples are included without performing the 
reverse transcription procedure. This control RNA preparation undergoes 
the same PCR.TM. process with the sample from the RT product. 
4. In Situ Hybridization 
Cells are treated with 0.25% trypsin for 5 min at 37.degree. C. The cell 
suspensions are transferred to poly-L-lysine-coated slides which are fixed 
with 4% paraformaldehyde for 20 min. After washing with PBS, the slides 
are dried with increasing gradients of ethanol and store at -80.degree. C. 
for future in situ hybridization. Rats are perfused intracardially with 
120 ml of saline at 40 ml/min, followed with 200 ml of fixative A (0.8 g 
NaOH, 8 g paraformaldehyde, 1.64 g sodium acetate in 200 ml distilled 
H.sub.2 O, pH 6.5) at 20 ml/min, followed by fixative B (1.4 g NaOH, 14 g 
paraformaldehyde, 13.35 g borax, pH 9.5). Brains are cryo-protected in 10% 
sucrose-fixative overnight at 4.degree. C. Coronal sections (15.mu.) are 
prepared and mount on subbed poly-L-lysine-coated slides and store at 
-80.degree. C. for in situ hybridization. 
Slides are dried overnight and subjected to 0.001% proteinase K digestion 
at 37.degree. C. for 20 min, then immersed in 0.1 M triethanolamine (TEA) 
with 0.25% acid anhydride for 10 min. Subsequent dehydration is carried 
out sequentially in 50, 70, 95 and 100% ethanol (3 min each). The 
hybridization is performed with a .sup.33 P-labeled cRNA probe (107 
cpm/ml) overnight at 55.degree. C. The cRNA probe is obtained from the 
cDNA clone in pKS vector using T7 or T3 RNA polymerase (Simmons et al., 
1989). After hybridization, the slides are washed sequentially in 
2.times., 1.times., 0.2.times., 0.1.times.SSC at 43.degree. C. and 
dehydrated in 50%, 70%, 95%, and 100% ethanol (3 min each). Brain slides 
are exposed to Kodak (Rochester, N.Y.) XAR-5 film overnight. After film 
development, the slides are processed by emulsion autoradiography. After 
emulsion film development, slides are counterstained with hematoxylin. 
After dehydration and mounting, slides are examined by dark-field 
microscopy. 
Importantly, the inventors have subjected in situ hybridization slides to 
DNAse treatment to exclude possible hybridization between the antisense 
probe and the plasmid cDNA construct. 
5. Protein Assay 
Proteins are determined using antibodies for NGF, p75.sup.NGFR, 
p140.sup.trkA, p145.sup.trkB (Hutton et al., 1992), and a monoclonal 
antibody (clone 27/21) specific for rat and mouse .beta. (2.5S) NGF that 
is also suitable for ELISA determinations if NGF proteins in brain tissue. 
Receptor antibodies are made to synthetic peptide segments on the 
different neurotrophin receptors and allow determination of different trks 
as well as differential staining of full and truncated versions of the 
different trks. 
6. Immunohistochemistry 
Cells are fixed in tissue culture wells by the gentle 1:1 addition of 8% 
paraformaldehyde in 0.12 M PBS (pH 7.3) for 20 min. After brief wash with 
PBS, the cells are ready to be immunostained. Rats are perfused 
intracardially with 120 ml of saline and 240 ml of a fixative solution 
containing 4% paraformaldehyde in 0.12 M phosphate buffered saline (PBS) 
(pH 7.3). Rat brains are removed and post fixed overnight at 4.degree. C. 
in 4% paraformaldehyde in 0.12 M phosphate buffer. Coronal sections 
(15.mu.) are prepared and mount on subbed poly-L-lysine coated slides. 
Cultures are washed with PBS buffer and incubated in normal goat serum (4%) 
for one hour. Following 3 5-min washes with PBS, the cultures are 
incubated with Goat anti-Mouse IgG (Chemicon International, Inc., 
Temecula, Calif.) antibodies (dilution 1:100) for two hours at room 
temperature. The cultures are quenched for endogenous peroxidase with 
0.03% of H.sub.2 O.sub.2 in methanol for 30 min at room temperature. After 
35-min washes with PBS, the cultures are incubated with Avidin-Peroxidase 
solution (Vector Lab, Burlingame, Calif.) for one hour at room 
temperature. Following 35-min washes with PBS, cultures are pre-incubated 
with diaminobenzidine tetrahydrochloride (DAB) (Vector Lab) and 0.006% 
H.sub.2 O.sub.2 for 10 min. 
Cultures are washed with distilled water and counterstained with 
hematoxylin. After dehydration and mounting, cultures are studied by light 
microscopy on an inverted (tissue culture) microscope. Similar procedures 
are used for p75.sup.NGFR, p140.sup.trkA and p145.sup.trkB receptor 
immunohistochemistry (Turner and Perez-Polo, 1993). For immunostaining of 
brain slices, coronal sections are mounted on slides and the same protocol 
is followed except that rinses are 5 times for 5-min each to minimize 
background staining. 
7. ELISA 
For in vitro studies, cells are treated with lysis buffer and extracted on 
ice for 10 min. The extractions are centrifuged at 20,000.times.g for 15 
min at 4.degree. C. The supernatant is assayed for protein using the 
Bradford method with a BSA standard (Ausubel et al., 1993). The samples 
are aliquoted and stored at -80.degree. C. for later ELISA. For in vivo 
studies, tissue from the hippocampus and other brain regions are dissected 
at 4.degree. C. and homogenized in ice-cold buffer (50 mM Tris; 2 mM EDTA; 
2 mM DTT; 100 .mu.M leupeptin; pH7.5) by a motorized Teflon pestle 
homogenizer (10 up-and-down strokes). The homogenates are then processed 
as described above for later ELISA. 
8. Antibody-Sandwich ELISA 
Specific anti-NGF or anti-BDNF antibodies are diluted to a final 
concentration of 0.2 to 10 .mu.g/ml. The optimal concentrations of 
anti-NGF or anti-BDNF antibodies and the conjugate necessary to detect the 
sample NGF or BDNF are determined by criss-cross serial dilution analysis. 
After final concentrations of anti-NGF or anti-BDNF antibodies are 
determined, wells of a plate are coated with serial concentrations of 
anti-NGF or anti-BDNF antibodies. The standard NGF and BDNF dilution 
series are prepared by 1:3 dilution of stock NGF and BDNF solutions. 
After tissue homogenization, sample homogenates are diluted in blocking 
buffer. 50 .mu.l aliquots of the diluted homogenate and standard NGF and 
BDNF dilutions are added to the antibody-coated wells and incubated two 
hours at room temperature. After washing the plate with water and blocking 
buffer, a 50 .mu.l antibody-alkaline phosphatase conjugate is added to 
each well and incubated for two hours at room temperature. After another 
wash with water and blocking buffer, 75 .mu.l of NPP substrate solution is 
added to each well followed by incubation for 1 hour at room temperature. 
After color development, the plate is read on a microtiter plate reader. 
The NGF and BDNF concentrations are determined from a standard 
concentration curve. 
9. Biological Assay of Neurotrophin Activity 
Biological assays for NGF induced differentiation are performed by assay of 
NGF activity in conditioned media, cells or tissues. Assays employ both 
the rat pheochromocytoma (PC12) cell line (Greene and Tischler, 1976) and 
nodose and dorsal root ganglia explants (Lindsay and Rohrer, 1985). The 
assays score neurite outgrowth and differentiation as seen in increased 
length and complexity of neurites. Assays employ standardization of 
neurotrophic dose-response curves as well as confirmation of blockade by 
appropriate antisera. PC12 cells are photographed and evaluated for 
morphological differentiation five days after treatment with appropriate 
experimental media. Nodose and dorsal root ganglia explants from embryonic 
day 8 are overlaid with the experimental media. 24-48 hours later, the 
ganglia are photographed and scored for neurite outgrowth (Perez-Polo, 
1987). 
10. Calculation of Transfection Efficiency 
Transformation efficiency in .beta.-Gal transfected cells is calculated 
after X-Gal staining and hematoxylin counterstaining by determining the 
total cell number and the number of .beta.-Gal positive cells in at least 
3 randomly chosen non-overlapping fields per well. Transformation 
efficiency is expressed as a percentage of .beta.-Gal positive cells to 
total cells. X-Gal staining offers a distinct advantage over other 
procedures that measure enzyme activity of the total population of 
transfected cells (e.g., luciferase-transfection quantitation assays) 
rather than enzyme activity within individual cells which can be 
visualized and counted. 
Transformation efficiency in NGF-and BDNF-transfected cells will first be 
estimated using quantitative RT-PCR.TM.. Studies have shown that such a 
qualitative comparison was sufficient for determining the relative optimal 
transformation efficiency when several cDNA:liposomes ratios are compared. 
In order to determine the precise transfection efficiency of an optimal 
ratio, in situ hybridization was combined with hematoxylin counterstaining 
to determine the total cell number and the number of successfully 
transfected cells (cells expressing NGF and BDNF mRNA at greater then 
background levels) in at least 3 randomly chosen non-overlapping fields 
per well. Transformation efficiency is expressed as a percentage of 
successfully transfected cells to total cells. 
Transfection efficiency for in vivo studies is calculated on the basis of 
cell counts made in 2.0 mm.sup.2 area units of coronal tissue sections 
adjacent to the site of injection. The total area sampled depends upon the 
extent of transfection observed. As stated for in vitro studies, 
.beta.-Gal transfection efficiency studies employ X-Gal staining and 
hematoxylin counterstaining to determine the total cell number and the 
number of .beta.-Gal positive cells. In studies of NGF and BDNF, in situ 
hybridization is combined with hematoxylin counterstaining to determine 
the total number and the number of successfully transfected cells. 
Transformation efficiency is expressed as a percentage of successfully 
transfected cells to total cells. 
11. Histopathological Assessment 
In general, histological assessments are done in conjunction with in situ 
and immunohistochemical analyses. Hemorrhage and necrosis is detected by 
conventional hematoxylin and eosin (H&E) staining. Edema formation 
(extravasated blood plasma) is monitored by immunostaining for plasma 
albumin, and then comparing this pattern to the hemorrhage in the adjacent 
serial H&E-stained section. Viable neurons in brain grey matter are 
visualized by conventional Nissel stain. Axonal injury is determined by 
Palmgren's silver impregnation method for the presence of reactive axonal 
swellings (retraction balls). 
Macrophages are detected using commercially-available monoclonal antibodies 
(ED1, ED2, ED3) specific for rat. Astroglial reaction is determined using 
Glial fibrillary acidic protein (GFAP) employing a mix of 
commercially-available mouse monoclonal antibodies. 
All of the compositions and methods disclosed and claimed herein can be 
made and executed without undue experimentation in light of the present 
disclosure. While the compositions and methods of this invention have been 
described in terms of preferred embodiments, it will be apparent to those 
of skill in the art that variations may be applied to the composition, 
methods and in the steps or in the sequence of steps of the method 
described herein without departing from the concept, spirit and scope of 
the invention. More specifically, it will be apparent that certain agents 
which are both chemically and physiologically related may be substituted 
for the agents described herein while the same or similar results would be 
achieved. All such similar substitutes and modifications apparent to those 
skilled in the art are deemed to be within the spirit, scope and concept 
of the invention as defined by the appended claims. 
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