40 CNS & Neurological Disorders - Drug Targets, 2012, 11, 40-49

Cerebrolysin, a Mixture of Neurotrophic Factors Induces Marked

Neuroprotection in Spinal Cord Injury Following Intoxication of
Engineered Nanoparticles from Metals
Preeti Kumaran Menon1, Dafin Fior Muresanu2, Aruna Sharma1, Herbert Mössler3 and
Hari Shanker Sharma*,1

1
Laboratory of Cerebrovascular Research, Department of Surgical Sciences, Anesthesiology and Intensive Care
Medicine, University Hospital, Uppsala University, SE-75185 Uppsala, Sweden
2
Department of Neurosciences, University of Medicine and Pharmacy “Iuliu Hatieganu”, Cluj-Napoca, Romania
3
Ever NeuroPharma, Oberburgau, Austria

Abstract: Spinal cord injury (SCI) is the world’s most disastrous disease for which there is no effective treatment till today.
Several studies suggest that nanoparticles could adversely influence the pathology of SCI and thereby alter the efficacy of
many neuroprotective agents. Thus, there is an urgent need to find suitable therapeutic agents that could minimize cord
pathology following trauma upon nanoparticle intoxication. Our laboratory has been engaged for the last 7 years in finding
suitable therapeutic strategies that could equally reduce cord pathology in normal and in nanoparticle-treated animal models
of SCI. We observed that engineered nanoparticles from metals e.g., aluminum (Al), silver (Ag) and copper (Cu) (50-60
nm) when administered in rats daily for 7 days (50 mg/kg, i.p.) resulted in exacerbation of cord pathology after trauma that
correlated well with breakdown of the blood-spinal cord barrier (BSCB) to serum proteins. The entry of plasma proteins
into the cord leads to edema formation and neuronal damage. Thus, future drugs should be designed in such a way to be
effective even when the SCI is influenced by nanoparticles. Previous research suggests that a suitable combination of
neurotrophic factors could induce marked neuroprotection in SCI in normal animals. Thus, we examined the effects of a
new drug; cerebrolysin that is a mixture of different neurotrophic factors e.g., brain-derived neurotrophic factor (BDNF),
glial cell line derived neurotrophic factor (GDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF) and
other peptide fragments to treat normal or nanoparticle-treated rats after SCI. Our observations showed that cerebrolysin
(2.5 ml/kg, i.v.) before SCI resulted in good neuroprotection in normal animals, whereas nanoparticle-treated rats required a
higher dose of the drug (5.0 ml/kg, i.v.) to induce comparable neuroprotection in the cord after SCI. Cerebrolysin also
reduced spinal cord water content, leakage of plasma proteins and the number of injured neurons. This indicates that
cerebrolysin in higher doses could be a good candidate for treating SCI cases following nanoparticle intoxication. The
possible mechanisms and functional significance of these findings are discussed in this review.
Keywords: Spinal cord injury, nanoparticles, silver, copper, aluminum, cerebrolysin, neurotrophic factors, brain-derived neurotrophic factor, glial cell
line derived neurotrophic factor, ciliary neurotrophic factor, blood-spinal cord barrier, spinal cord edema, neuronal injuries, spinal cord pathology.

1. INTRODUCTION
Spinal cord injury (SCI) is a debilitating disease that occurs
following motor vehicle accidents or falls and inflicts damage to cells
within the spinal cord [1-6]. Depending on the magnitude and
severity, SCI could result in quadriplegia/tetraplegia, paraplegia and
other lifetime disabilities [7-10]. Thus, efforts should be made to treat
SCI victims in time with suitable drugs to restore functioning of the
spinal cord in order to improve the quality of life. SCI can either be
traumatic or non – traumatic [11]. Traumatic injuries are caused by
various factors such as road traffic accidents, domestic work-related
accidents, sports injuries and gunshot or knife wounds [5, 6, 10, 11].
Non- traumatic injuries can be caused by infection of the nerve cells
present in the spinal cord and cysts or tumors [4-6]. SCI can also be
described as complete or incomplete, depending on the complete or
partial loss of function below the point of injury [5, 6, 9-11].
1.1. Vascular Events Contributing to Secondary Pathogenesis
Injury to the spinal cord first results in mechanical disruption of
spinal cord structures, known as primary injury that slowly
*Address correspondence to this author at the Laboratory of Cerebrovascular
Research, Department of Surgical Sciences, Anesthesiology and Intensive
Care Medicine, University Hospital, Uppsala University, SE-75185
Uppsala, Sweden; Tel/Fax: +46 18 24 3899; E-mail: Sharma@surgsci.uu.se
progresses into secondary injury by damaging cells in the grey and
white matter of the cord [5, 6, 9-11]. The primary injury results in
disruption of the blood vessels [5, 11]. There is a decrease in blood
flow to the damaged tissue and it results in the poor delivery of
oxygen and nutrients to the neurons [9-11].
Secondary pathogenesis includes restriction in blood flow,
excessive release of neurotransmitters, inflammation created by
immune cells, free radical production and self-destruction of nerve
cells [9-13]. All of these mechanisms result in the increase in the
area of damage within the spinal cord [5, 6, 9-13]. The axons are
damaged and then there is formation of a glial cell scar [see 5, 6,
11]. However, it remains unclear how the nerve cells destroy
themselves after injury and how the neurons are flooded with
excitatory neurotransmitters such as glutamate [14].
1.2. Blood-Spinal Cord Barrier Breakdown
Under normal conditions the blood-brain barrier (BBB) and
blood-spinal cord barrier (BSCB) do not allow the passage of large
molecules into the central nervous system (CNS) (Fig. 1) [13, 14].
The BBB and BSCB contain specialized endothelial cells that help
in strict regulation of the micro-fluid environment of the CNS [7, 8,
14]. The tight junctions that are present between these adjacent
endothelial cells help in preventing large molecules such as plasma
proteins from entering the neuropil (Fig. 1) [7, 8, 13]. The
glycocalyx, which is rich in glycoproteins, is located on the

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Cerebrolysin, a Mixture of Neurotrophic Factors in SCI CNS & Neurological Disorders - Drug Targets, 2012, Vol. 11, No. 1 41

Fig. (1). Structure and function of the blood-spinal cord barrier (BSCB). A. Schematic diagram showing endothelial cells (E) in the spinal cord connected with
tight junctions and surrounded by a thick basement membrane. The glial cells (G) and nerve cells (N) around the spinal cord endothelial cells are clearly seen.
The glial cell covering of the spinal cord microvessels is less intense particularly in large diameter microvessels [for details see Sharma 2004, Ref. 14].
Intravascular tracer is normally stopped at the tight junction and does not penetrate the luminal endothelial cell membrane to reach extracellular space
indicating a very tight BSCB (A). Likewise, intrathecal tracer does not normally pass the abluminal endothelial cell membrane and/or the tight junctions to
reach the vascular compartment (B), suggesting that the BSCB effectively regulates exchange of substances between spinal cord microenvironment and the
vascular system (B). Data modified after [14]. C. However, essential nutrients and waste materials could cross the BSCB freely from blood to spinal cord and
vice versa. D. BSCB showing the relationship between neurons (N), glial cells (G), endothelial cells. Lanthanum (La) in the lumen is stopped at the tight
junction (TJ). The capillary is surrounded by a thick basement membrane (BM). Data modified after Sharma 2004 [14].

endothelial cells. It has a negative charge and thus blocks plasma [11-15]. Therefore, the BBB or BSCB structures help in the normal
proteins because of their similar or charge [15, 16]. The basement functioning of neurons [7, 8].
membrane, which is present on the parenchymal side of the Following injury to the brain or spinal cord the BBB elctrostatic
endothelial cell, provides structural support to these cell membranes BSCB are disturbed and there is a rapid entry of albumin into the
42 CNS & Neurological Disorders - Drug Targets, 2012, Vol. 11, No. 1
brain or spinal cord compartment [7-11, 14]. The presence of
albumin inside the neuropil, neurons or non-neuronal cells can be
identified using albumin immunohistochemistry [17, 18]. The brain
or spinal cord is then exposed to the various immune system cells
such as neutrophils, T-cells, macrophages and monocytes. These
immune cells produce an inflammatory response that results in
damage to nerve cells. In certain types of injury, however, these
immune cells prove to be protective and it is still controversial
weather the immune system is protective or destructive [19-21].
The entry of immune cells into the CNS results also in the increased
production of free radicals [21, 22]. These free radicals are highly
reactive form of oxygen molecules and cause destruction of
neurons. Once the BBB or BSCB is damaged free radicals could
cause neuronal damages [23-25].
1.3. Spinal Cord Edema Formation
Once the BBB or BSCB is disrupted and serum proteins enter
the extracellular environment of the CNS, there is a formation of
edema inside the brain or spinal cord [26-30]. Edema is caused by
an increase in the water content of the spinal cord or brain [31-33].
There are two main types of brain edema formation e.g., vasogenic
and cytotoxic [see 31]. Vasogenic edema occurs when plasma
proteins and water leak into the extracellular space of the brain.
Cytotoxic edema forms when water accumulates inside the
intracellular brain compartment [31-33]. During cytotoxic edema
there is a swelling of neuronal, non-neuronal, and endothelial cells
[31]. Vasogenic edema formation occurs due to an increased rate of
capillary filtration and reduced rate of removal of fluids from the
tissues [8, 11, 29-33].
There are several neurochemicals that can cause an alteration in
BBB or BSCB permeability and influence edema formation inside
the brain or spinal cord, thereby resulting in damage to cells and
tissues [26-33]. These neurochemicals are serotonin, prostaglandins
or histamine, among others [8-11, 14]. In many experimental cases
it has been proved that if these neurochemicals are inhibited before
SCI then it will help to attenuate BSCB disruption, edema
formation and/or cell injury [see 29-33].
In the clinical or experimental situations it has been observed
that plasma protein leakage and spread of edema fluid increases
with the advancement of time [31]. The severity of brain or spinal
cord damage influences the intensity of the edematous swelling [15-
18].
2. NEUROTROPHIC FACTORS IN SPINAL CORD INJURY
There are various neurotrophic factors such as nerve growth
factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary
neurotrophic factor (CNTF) and glial cell line derived neurotrophic
factor (GDNF) present into the CNS [34-36]. These growth factors
are required for the survival and proper functioning of neurons [11-
13]. When there is an injury in the cord immune cells release
cytokines, which in turn upregulate neurotrophic factors; their
production, however, is not sufficient and as a result it neither
prevents the degeneration of neurons nor promotes their
regeneration [see 13, 27, 28, 33-36].
In many experimental cases these growth factors have been
administered exogenously over the injured cord that resulted in
reducing the pathological outcome [13, 33-36]. When BDNF or
NGF was administered alone in the early phase of injury they
improved the conditions of the injured cord by minimizing BSCB
disruption and edema formation [see 13]. However, if they were
given at 60-90 minutes after the injury then there was no sign of
improvement [33-36].
When BDNF or GDNF was administered in combination over
the injured cord at 60-90 minutes after injury they attenuated the
pathological conditions of the cord by reducing BSCB disruption
Menon et al.
and edema formation [13]. Certain combinations of neurotrophic
factors when administered exogenously at 30-90 minutes after
injury did not prove to be effective, for example BDNF with NGF
or neurotrophin-3 [13]. Therefore, the combinations of neurotrophic
factors should be made in such a way that they produce useful
biological effects [33-36].
Various studies showed that the time of application and dose of
various neurotrophic factors depends on their receptor expression
level that changes with different phases after injury [11-13]. A high
concentration of neurotrophic factors when given in combination
improves motor function and helps in the regeneration of neurons
[33-36]. Sometimes spinal cord injury also decreases the level of
growth hormones. Therefore they are administered in high
concentrations exogenously to provide some neuroprotective effects
[37, 38].
3. NANOPARTICLES INFLUENCE PATHOLOGY OF
SPINAL CORD INJURY
In the environment where we live, we have different types of
nanoparticles and this has recently attracted the attention of various
researchers in the field of SCI to explore how these nanoparticles
may influence the pathophysiology of CNS injury [39-46].
Nanoparticles, which are considered to be a recent research area,
can prove to be either constructive or destructive [46]. When they
are sized in the range of 50-200 nm they can be used for various
biomedical applications such as for diagnostic and treatment
purposes but sometimes they can produce some toxic effects [47,
48]. However it is still not clear how nanoparticles produce
neurotoxicity.
Recent experiments suggest that in an environment where
nanoparticles are abundant the extent of neural injury can be
exacerbated as, for example, soldiers in a combat setting who are
exposed to different kinds of nanoparticles such as sulphur, copper,
silica or carbon. In this situation, the pathophysiology of spinal cord
injury is likely to differ from from persons who receive SCI in a
clean and healthy environment [40-46]. However, details of these
conditions are still not well known.
Nanoparticles inhaled from the environment can enter the body
fluid system and then through endocytosis enter various neuronal
and non-neuronal cells and induce cellular toxicity [see 46]. When
the cells or tissues are exposed to these small particles in the size
range of 10-100 nm, they exert higher inflammation [2, 46].
3.1. Exposure of Nanoparticles Results in BSCB Damage
When nanoparticles such as silver, copper or aluminum in the
size range of 50-60 nm were administered systematically, it induces
the breakdown of BSCB thereby making it permeable to various
protein tracers such as Evans blue albumin and radioiodine [39, 42].
Intravenous or intracerebral administration of these nanoparticles
can result in severe damage of the BBB and BSCB whereas
intraperitoneal administration results in less damage [41, 42].
Further investigations are required in order to study how the dose of
nanoparticles and their route of administration result in the damage
of BSCB.
3.2. Nanoparticles Results in the Formation of Edema
Nanoparticles also exacerbate edema formation [44, 45]. The
edema formation in terms of an increase in brain water content was
more than 1% when the nanoparticles were administered
intravenously [41-46]. Water content inside the brain is measured
as the difference between dry and wet weight of the sample [45].
Obviously, BSCB disruption and permeability to various
protein tracers leads to the formation of vasogenic edema [1-6].
BSCB disruption and edema formation then result in morphological
changes inside the brain [39-46]. This indicates that the
nanoparticles also provoke damage to various cells and tissues.
Cerebrolysin, a Mixture of Neurotrophic Factors in SCI CNS & Neurological Disorders - Drug Targets, 2012, Vol. 11, No. 1 43

4. TREATMENT STRATEGIES FOR SCI (d) Neurogenesis- it allows the formation of neurons by
The initial response of the spinal cord to injury is a rapid differentiation of progenitor cells.
swelling [5-8]. Therefore the injured patient requires immediate Due to these mechanisms, cerebrolysin is considered as a
treatment within 4 to 8 hours of primary injury to maximize the multimodal drug that has an immediate neurotrophic activity [49-53].
chance for recovery [11-14]. Normally, the patient is treated with So far cerebrolysin has been an effective therapeutic treatment
three different types of drugs, e.g., anti-inflammatory compounds for CNS injuries. However, its role in SCI and related disorders is
that target immune responses, anti-oxidants drugs that reduce free still under investigation.
radical formation, and anti-excitotoxins that prevent the effects of
excessive release of excitatory neurotransmitters such as glutamate 6. OUR INVESTIGATIONS OF CEREBROLYSIN IN SCI
[see 5, 11, 14].
We examined effects of nanoparticles on the pathophysiology
Methylprednisolone is an anti-inflammatory drug that is of SCI in our rat model (Fig. 2). Furthermore the potential efficacy
effective only if administered within 8 hours of injury [11]. This of cerebrolysin was examined in normal or nanoparticle-treated rats
drug responds to the injury by reducing immune system responses. after SCI.
However, there are various limitations of this drug, which cannot be
ignored [11, 14]. In cases where the SCI is severe enough to induce
complete paralysis then higher doses of drug are given. However, if
methylprednisolone is given in higher doses then it can result in
several complications such as muscle weakness, blood clot in veins
and/or no improvement in the cell injury [11].
Lazaroids, a class of 21-aminosteroids, are anti-oxidant drugs
and they respond to the injury by preventing the excessive
production of free radicals [1-4, 11, 14]. They are modified from
methylprednisolone, and the U.S. Food and Drug Administration
has not approved their use as they are created from the existing
unapproved methylprednisolone drug [11]. The efficacy of these
drugs in SCI is still under investigation.
Thyrotropin-releasing hormone is an anti-excitotoxin and
minimizes the excessive production of neurotransmitters such as
glutamate by releasing thyroid-stimulating hormones. However,
further research is required for the drug in SCI [11, 14].
These drugs work in normal SCI patients but they do not seem
to be effective in cases where spinal cord injury is exacerbated due
to nanoparticle exposure [9-14, 39-46]. Therefore, the drug should
be designed in such a way that it enhances neuroprotection in both
normal SCI and in nanoparticles-influenced injury.

5. CEREBROLYSIN: A PROMISING DRUG FOR THE

TREATMENT OF SCI
Cerebrolysin is the only drug available for clinical use
containing active fragments of some important neurotrophic factors
[49-53]. This drug is used to induce neuroprotection when there is
an injury in the CNS and also helps in the regeneration of neurons
in neurodegenerative diseases like multiple sclerosis, Parkinson’s
disease, Alzheimer’s disease, dementia, and acute or chronic stroke
[see 39, 49-53]. It is the only drug whose action is similar to the
various neurotrophic factors in combination such as BDNF, GDNF,
CNTF and NGF and others [see 39]. If a higher dose of this drug is
administered systematically then it proves to be effective in the
situation where injury in the spinal cord is influenced by exposure
to nanoparticles, but further research is still required [39, 49-55].
5.1. Possible Mechanisms of Cerebrolysin-Induced
Neuroproetction
There are various mechanisms of cerebrolysin induced
neuroprotection [see 39]:
(a) Neuronal survival - The neurotrophic factors that are
present in this drug helps in the survival of neurons and
prevents the cell death.
(b) Neuroprotection-It induces neuroprotection inside the brain
even in the detrimental conditions by maintaining the
cellular interactions.
(c) Neuroplasticity –It helps the process of re-wiring thereby
allowing the brain to adapt to various changes in the
environment. It also helps in the sprouting of axons or
dendrites.
Fig. (2). Spinal cord injury model in rats. A longotudonal incision is made into
the right dorsal horn of the T10-11 segments (L). The lesion dimensions are
5
mm long and
2 mm deep (b). The deepest portion of the lesion is largely limited
to the Rexed’s laminae VII (a). Tissue specimen examined for morphology or
biochemistry was collected from the rostral (T9) and caudal (T12) segments of
the lesion. Data modified after Sharma 2004 [14]. Bar = 5 mm.
6.1. SCI Model
Under equithesin anesthesia (3 ml/kg, intraperitoneal), the
injury was inflicted by making a longitudinal incision (1.5 mm deep
and 4 mm long) into the right dorsal horn of the T10-11 segment of
the spinal cord in male Wistar rats (Fig. 2) [54-56]. All experiments
were approved by the local institutional ethics committee and
conducted according to the National Institutes of Health guidelines
for the care of experimental animals [57].
6.2. Nanoparticle Administration
Engineered nanoparticles from Cu, Ag or Al in the size range of
50-60 nm (obtained from US Wright Patterson Air Force Research
Base, Dayton, OH, USA), were first suspended in 0.05% Tween 80
solution and then administered intraperitoneally (50 mg/kg once
daily) for seven days [39, 45].
6.3. Treatment with Cerebrolysin
Cerebrolysin (Ever Neuro Pharma, Austria) at a dose of either
2.5 ml/kg or 5.0 ml/kg was administered intravenously in rats 30
44 CNS & Neurological Disorders - Drug Targets, 2012, Vol. 11, No. 1 Menon et al.

Fig. (3). Representative examples of the leakage of serum albumin in the spinal cord and its modification by nanoparticles and cerebrolysin treatment. SCI
alone resulted in massive leakage of albumin within the neuropil of the spinal cord that is seen around neurons or microvessels (arrows). Deposit of albumin in
perineuronal and perivascular area represents edematous swelling that could lead to cell and tissue injury (c). This albumin leakage was exacerbated after SCI
by Ag (b) or (Cu) treatment. Cerebrolysin (2.5 ml) (d) did not markedly reduce Ag nanoparticle-induced exacerbation of albumin leakage following SCI.
Although cerebrolysin (2.5 ml) considerably reduced albumin leakage in normal rats after SCI (h), a high dose (5 ml) significantly recued nanoparticle-
induced exacerbation of albumin extravasation in SCI in both Ag (a) and Cu (e) treated groups. Bar = 30
m (Sharma et al., unpublished observations).

minutes before SCI in saline or nanoparticle-treated animals. The
animals were allowed to survive 5 hours after injury [39, 49-53].
Normal rats were used as controls [58].
6.4. Nanoparticle Effect on BSCB Permeability
The BSCB permeability was quantified with Evans blue
albumin (EBA) leakage (2% of 3 ml/kg EBA intravenous) and
serum albumin immunohistochemistry. EBA leakage was measured
in the tissue calorimetrically [45, 52-55]. Leakage of albumin was
demonstrated immunohistochemically using a primary albumin
antibody (1:4000) on paraffin sections from the spinal cord. The
immunoreaction was developed using peroxidase-antiperoxidase
technique [17, 18]. Nanoparticle-treated animals subjected to SCI
exhibited BSCB breakdown that was much higher in terms of
Evans blue or albumin leakage than the control group after trauma
(Fig. 3, Table 1). This effect was most pronounced with Ag and Cu
nanoparticles [43, 44].
6.5. Nanoparticles Affect Spinal Cord Edema Formation
Spinal cord water content was used to determine edema
formation and calculated as the difference between the wet and dry
weights of the tissues sample [55]. We observed albumin leakage
into the spinal cord suggesting that serum proteins, when entering
the cord could result in edema formation [54-56]. This was further
confirmed by the measurement of water content that was evaluated
in the nanoparticle- or saline-treated animals after SCI [55]. The
nanoparticle-treated animals without injury showed a mild increase
in water content whereas in nanoparticle-treated rats there was a
significant increase in water content after SCI (Table 1). Saline-
treated rats also showed spinal cord edema but the magnitude was
much less than a combination of nanoparticles and trauma. Ag and
Cu exerted the most pronounced effects on edema formation after
injury compared to Al nanoparticles [44, 45].

Table 1. Effect of Various Nanoparticles on Pathology of Spinal Cord Injury in Rats

EBA (mg%) Spinal Cord Water (%) Neuronal Damage (nr)

Expt. Type
T9 T12 T9 T12 T9 T12

Control 0.23±0.04 0.25±0.06 64.23±0.12 64.56±0.08 2±1 1±2

Ag 0.42±0.08** 0.44±0.06** 65.89±0.21** 65.84±0.21** 8±2** 8±4**
Cu 0.46±0.06** 0.48±0.05** 65.94±0.13** 65.89±0.10** 10±4** 9±3**
Al Treated 0.40±0.08* 0.42±0.06** 65.13±0.07* 65.23±0.10* 8±2** 7±2*
SCI 0.94±0.12**a 0.98±0.14**a 66.23±0.12**a 66.54±0.21**a 15±3**a 18±6**a
Ag 1.89±0.14**b 1.98±0.23**b 68.34±0.43**b 68.89±0.28**b 36 ± 8**b 43±6**b
Cu 1.98±0.18**b 2.04±0.21**b 68.89±0.34**b 68.94±0.33**b 44±7**b 48±9**b
Al Treated 1.24±0.08*b 1.43±0.10*b 67.56±0.12*b 67.78±0.23*b 28±8*b 24±6*b
* P <0.05, ** P <0.01 from control, a = significantly different from control, b = Significantly different from SCI ANOVA followed by Dunnett’s test for multiple group comparison
from one control. Data are means±SE of 5 to 6 rats at each point. EBA, Evans blue albumin; SCI, spinal cord injury.
Cerebrolysin, a Mixture of Neurotrophic Factors in SCI CNS & Neurological Disorders - Drug Targets, 2012, Vol. 11, No. 1 45

6.5. Nanoparticles Induce Morphological Changes Inside the
Cord
Morphological changes such as neuronal or glial cell damage in
control, injured and cerebrolysin-treated animals after SCI were
observed by various standard histological and immunostaining
techniques such as Nissl or Haematoxylin and Eosin (HE) stain for
neuronal injury and glial fibrillary acidic protein (GFAP)
immunostaining for astrocytic reactions [17, 18, 39, 45-52].
To observe the neuronal or glial cell changes inside the spinal cord,
4 μm thick sections were cut and then stained for GFAP, Nissl or HE
[17]. The nanoparticle-treated animals before and after injury both
showed neuronal cell damage (Figs. 3-5). However, nanoparticle-
treated animals after injury showed more damaged neurons and
astrocytes in comparison to before the injury, where there was only a
mild neuronal or glial reaction (Table 1, Figs. 4, 5).
6.6. Effect of Cerebrolysin on Spinal Cord Pathophysiology
Effect of cerebrolysin was examined in three groups of rats.
The first (control) group did not receive nanoparticle administration
nor was subjected to any kind of SCI. The second group was
subjected to SCI only, and the third group received nanoparticles
and SCI. Two doses of cerebrolysin (2.5 ml/kg and 5.0 ml/kg) were
administered intravenously 30 minutes before the injury. The lower

Fig. (4). Representative examples of neuronal cell damage in the spinal cord and its modification by nanoparticles and cerebrolysin treatment. SCI resulted in
marked neuronal damage in the normal rat (c) and this neuronal injury was further aggravated by Ag (d), Cu (h) and Al (g) nanoparticle treatment. Neuronal
cell damage (arrows) appears to be exacerbated after SCI in Ag and Cu treated rats compared to Al. Cerebrolysin treatment (2.5 ml) reduced neuronal injury
after SCI in normal rats (b), but failed to do so in Ag-treated spinal cord traumatized rats (f). On the other hand high dose cerebrolysin (5 ml) markedly
reduced this exacerbation of neuronal damage in SCI by Ag (a) or Cu (e) treatment. Bar = 30
m. Haematoxylin & eosin tain on 3-4
m thick paraffin
embedded spinal cord sections (Sharma et al., unpublished observations).

Fig. (5). Representative examples of glial cell damage in the spinal cord and its modification by nanoparticles and cerebrolysin with or without nanowired
delivery. Nanowired cerebrolysin (NWCBL) was administered in spinal cord injured group and the results are compared with normal cerebrolysin (CBL)
delivery in identical dosage on astrocytic expression examined by glial fibrillary acidic protein (GFAP) immunoreactivity on paraffin sections. The results
showed that NWCBL is able to thwart activation of GFAP in SCI after nanoparticle intoxication. Five h SCI resulted in marked activation of GFAP in the
spinal cord (a). Pretreatment with Ag nanoparticles exacerbated this astrocytic activation after SCI (b). Normal cerebrolysin given in 2.5 ml dose in Ag (c) or
Cu (f) treated spinal cord inured rats did not reduce GFAP activation markedly. However, NWCBL given in comparable doses markedly reduced GFAP
activation in Ag (e) or Cu (d) treated rats after SCI. The activation of GFAP is largely seen in SCI around neurons or blood vessels (Arrows, arrow heads). Bar
= 25
m (Sharma et al., unpublished observations).
46 CNS & Neurological Disorders - Drug Targets, 2012, Vol. 11, No. 1 Menon et al.

Table 2. Effects of Cerebrolysin on Pathology of Spinal Cord Injury in Rats

EBA (mg %) Spinal Cord Water (%) Neuronal Damage (nr)

Expt.Type
T9 T12 T9 T12 T9 T12

A. Control
2.5 ml/kg Cerebrolysin 0.24±0.04 0.25±0.041 65.13±0.12 65.10±0.03 1±1 nil
5.0 ml/kg Cerebrolysin 0.20±0.03 0.18±0.08 65.08±0.10 65.10±0.02 nil nil
B. Normal SCI
2.5 ml/kg Cerebrolysin 0.54±0.06* 0.58±0.10* 65.34±0.12* 65.44±0.14* 3±2* 2±1*
5.0 ml/kg Cerebrolysin 0.34±0.08a 0.36±0.04a 65.10±0.07a 65.06±0.04a 2±2* 1±2*
C. Nanoparticles+ SCI
2.5 ml/kg Cerebrolysin
Ag+SCI 1.04±0.11a 1.14±0.17a 67.89±0.12a 67.67±0.32a 23±6a 22±8a
Cu+SCI 1.21±0.12a 1.34±0.21a 67.76±0.34a 67.88±0.23a 20±6a 19±5a
Al+SCI 0.89±0.21a 0.94±0.22a 67.06±0.10a 66.89±0.21a 18±6a 15±3a
5.0 ml/kg Cerebrolysin
Ag+SCI 0.67±0.10b 0.65±0.12b 65.34±0.12b 65.04±0.14b 8±3b 7±4b
Cu+SCI 0.74±0.14b 0.68±0.14b 65.05±0.08b 65.34±0.18b 7±6b 8±2b
Al+SCI 0.68±0.08b 0.72±0.10b 65.13±0.21b 65.06±0.21b 4±6b 3±4b
* P <0.05, a = significantly different from control, b = Significantly different from SCI. ANOVA followed by Dunnett’s test for multiple group comparison from one control. Data
are means±SE of 5 to 6 rats at each point. EBA, Evans blue albumin; SCI, spinal cord injury.

dose of cerebrolysin had moderate effects in reducing the brain

water content whereas the higher dose had greater effects in
reducing the water content in all the three cases (Table 2, Figs. 3-5).
There was a moderate decrease in BSCB permeability with the
lower dose of cerebrolysin whereas the higher dose of cerebrolysin
attenuated EBA leakage, edema formation and neuronal cell
damage in the nanoparticles-treated group (Table 2). In some group
TiO2 nanowired cerebrolysin in SCI was also examined (see Fig.
5).
The higher and lower doses of cerebrolysin both resulted in
reduction of neuronal and glial cell damage following SCI in
normal rats but the higher dose of cerebrolysin or nanowired
cerebrolysin was needed to significantly reduce edema formation,
BSCB breakdown and neuronal cell injury after SCI in
nanoparticle-treated animals as compared to the lower doses in this
group (Table 2).

7. POSSIBLE MECHANISMS OF CEREBROLYSIN

INDUCED NEUROPROTECTION IN SCI
We observed that the nanoparticles (50-60nm) when
administered intraperitoneally to the animals followed by SCI can
result in exacerbation of BSCB breakdown. The leakage of plasma
proteins into the spinal cord can result in the formation of edema,
increased water content inside the cord leading to severe neuronal
and non-neuronal cell reactions (Fig. 6).
Our results further show that cerebrolysin in low or high doses
resulted in marked reduction in neural injury, glial cell activation,
reduction in BSCB breakdown and edema formation in normal rats
after SCI. However, in nanoparticle-treated rats after SCI, a high
dose of cerebrolysin was needed to achieve neuroprotection. This
means that nanoparticle treatment by itself could induce marked
depletion of neurotrophic factors in the cord. Thus, imposing SCI to
these nanoparticle-treated animals appears to increase the amount
of neurotrophin replacement needed. As a result, low dose
cerebrolysin was ineffective in reducing cell injury in nanoparticle-
treated and injured rats. Obviously, high dose cerebrolysin could
supply the required amount of neurotrophins to counteract cell
injury.
Fig. (6). Schematic diagram of spinal cord or brain injury induced cell
damage. It appears that breakdown of the BBB or BSCB is instrumental in
precipitating neuronal damage due to edema formation and alterations in
gene expression. Our results showed that cerebrolysin restores the
endothelial cell membrane structure and function and thus potentiates BSCB
or BBB function in SCI. Obviously, strengthening of the BBB or BSCB will
result in less edema formation and neuronal injury [for details see 7].
There are many neurotrophic factors such as BDNF, GDNF,
CNTF or NGF, which can be used to treat normal injuries, but in
the case of SCI where it is influenced by nanoparticles these
neurotrophic factors are not so effective (results not shown). Our
investigations show that cerebrolysin, which contains a
combination of various neurotrophic factors, amino acids, vitamins,
macronutrients, micronutrients and anti-oxidant enzymes could
definitely induce neuroprotection [39, 49-52]. Thus, as mentioned
Cerebrolysin, a Mixture of Neurotrophic Factors in SCI CNS & Neurological Disorders - Drug Targets, 2012, Vol. 11, No. 1
above, higher dose of this drug was effective in the case where CONFLICT OF INTEREST
nanoparticles were administered to the animals prior to trauma [see The authors have no conflict of interest with any organizations
39]. On the other hand, a lower dose of this drug worked effectively or entities listed above.
in normal SCI. Cerebrolysin resulted in significant decrease in
BSCB permeability, reduction in water content and reduced the ABBREVIATIONS
extent of neuronal or glial cell damage inside the cord. If this drug
is administered after 1- 2 hours after injury it does not result in any SCI
significant reduction of cord pathology (results not shown). BBB
Therefore, cerebrolysin induces neuroprotection inside the cord
depending on its dose and time [39, 49-52]. We observed dose- CNS
related effects of cerebrolysin in inducing neuroprotection within NGF
the cord. So it is clear from the data that higher dose cerebrolysin BDNF
has good results when compared to the lower dose, especially in
nanoparticle-treated and injured rats. Further investigations are GDNF
needed with the time- related effects of this drug. CNTF
The probable mechanisms of nanoparticle-induced BSCB BSCB
disruption, edema formation and cellular injury are unclear. It
appears that neuronal nitric oxide synthase (nNOS) up-regulation EBA
could contribute to such injury caused by nanoparticles [42-45]. nNOS
Since SCI in itself induces nNOS expression [23-32], a
combination of nanoparticles and SCI will exacerbate nNOS REFERENCES
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However, this is a subject that requires further investigation.
8. CONCLUSION AND FUTURE PERSPECTIVES [2]
These results strongly suggest that nanoparticles aggravate SCI-
induced BSCB breakdown, edema formation and neuronal or glial
cell injury. Cerebrolysin appears to be very effective in reducing
pathophysiology of SCI in normal or nanoparticle-treated injured [3]
animals by strengthening BSCB function (Fig. 6) in comparison to
other neurotrophic factors. Thus, cerebrolysin has proved to be a
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being investigated in our laboratory.
[6]
ACKNOWLEDGEMENTS
The work reported here is partially supported by grants from the [7]
Air Force Office of Scientific Research (EOARD, London), and Air
Force Material Command, USAF, under grant number FA8655-05-
1-3065 for studies on biological effects of nanoparticles. Work on
the CNS injury and repair program is supported by Grants from
Swedish Medical Research Council (Nr 2710-HSS), Göran
Gustafsson Foundation, Stockholm, Sweden (HSS), Astra Zeneca, [8]
Mölndal, Sweden (HSS/AS), The University Grants Commission,
New Delhi, India (HSS/AS), Ministry of Science & Technology,
Govt. of India (HSS/AS), Indian Council of Medical Research,
New Delhi, India (HSS/AS), Amity Science & Technology
Foundation, New Delhi, India (AS/HSS), India-EU Co-operation [9]
Program (HSS/AS), US Food & Drug Administration (FDA),
National Center for Toxicological Research (NCTR), Jefferson,
AR, USA (PKM/HSS) and University of Arkansas Fayetteville, [10]
AR, USA (HSS), The Rockefeller Foundation, AR, USA (HSS).
The views expressed here are solely those of the authors and in no [11]
way reflect the official position of any of the granting organizations
or collaborating entities mentioned above. We thank Prof. Lars [12]
Wiklund, Dr. Thomas Winkler, Docent Peymann Björklund,
Uppsala University Hospital for extending their co-operation, help
and suggestions during the work and critically reading the [13]
manuscript. We are indebted to reviewers for valuable suggestions
that have improved the manuscript immensely. We thank Mari-
Anne Carlsson, Kärstin Flink, Ingmarie Olsson, Margareta Butler
[14]
and Ulla Johansson for technical assistance and help though the
experimental work and histopathology.
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Received: July 30, 2011 Revised: November 15, 2011
PMID: 22229324
CNS & Neurological Disorders - Drug Targets, 2012, Vol. 11, No. 1 49
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Accepted: December 15, 2011