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Stable gastric pentadecapeptide BPC 157 can improve the healing course of spinal cord injury and lead to functional recovery in rats
Introduction
We focused on the application of the stable gastric pentadecapeptide BPC 157 [1–11] to improve the outcomes
of spinal cord injury in rats.
Spinal cord injury generally involves the preclusion of
neural relays across the lesion site and is thereby predictably associated with a lack of functional improvement [12, 13]. On the other hand, there is evidence that
spinal cord injury triggers a cascade of secondary degenerative events that cause further damage to the injured
area and induce local inflammation along with
hemorrhage and edema [12, 13] and that the therapeutic
agents imatinib (which has been shown to inhibit cytokine production and reduce hemorrhage, edema, and inflammation) [14] and ibuprofen initiate favorable axonal
growth and functional recovery through Rho inhibition
[15]. Likewise, there is favorable evidence to support the
engraftment of neural stem cells [16] or bone marrow
stromal cells [17] into the lesion site. However, there are
disputes about the relevant applicability of this evidence
[18, 19], particularly considering the low survival rate of
bone marrow stromal cells transplanted into the contused adult rat spinal cord [20, 21] and the need to completely fill the lesion site with neural stem cells [22].
Consequently, there have been attempts to improve the
therapeutic effectiveness with combined treatments (i.e.,
neural stem cells with fibrin and a growth factor cocktail
(BDNF; NT-3; mGDNF; IGF; bFGF; EGF; PDF; aFGF;
and HGF) [23] or bone marrow stromal cells with the
application of cyclosporine, minocycline, and methylprednisolone [24]). Likewise, considering the beneficial
effect of the deletion of the Nogo Receptor 1 (NgR1)
gene, a sequential combination of Nogo-A suppression
(by anti-Nogo-A antibody treatment) and treadmill
rehabilitative training was examined [25].
It is generally believed that further attempts are fully
justified [26]. In comparison, the stable gastric pentadecapeptide BPC 157, an emerging treatment with potential therapeutic applications, appears to be unrestricted
by the limitations seen in previous therapies. The stable
gastric pentadecapeptide BPC 157, an original cytoprotective antiulcer peptide that is used in ulcerative colitis
and recently in a multiple sclerosis trial and that has an
LD1 that has not been achieved [1–11], is known to
have pleiotropic beneficial effects [1–11] and to interact
with several molecular pathways [2, 27–32]. BPC 157
has beneficial effects on inflammation, hemorrhage, and
edema after traumatic brain injury [33], various severe
encephalopathies (which follow gastrointestinal and/or
liver lesions), NSAID overdose [34–37], or insulin overdose seizures [38] and on severe muscle weakness after
exposure to the specific neurotoxin cuprizone in a rat
multiple sclerosis model [39] or magnesium overdose
[40]. In other studies, it was shown that BPC 157
counteracts increased levels of proinflammatory and
procachectic cytokines such as IL-6 and TNF-α [2].
Finally, BPC 157 improves sciatic nerve healing [41]
when applied intraperitoneally, intragastrically, or locally
at the site of anastomosis shortly after injury or directly
into the tube after non-anastomosed nerve tubing (7-
mm nerve segment resection).
Therefore, we used a model of spinal cord injury that
has many characteristics found in human spastic syndrome [42] and can be used long-term to provide a realistic model of spasticity development in the tail muscle.
The administered therapy was a one-time intraperitoneal application of the stable gastric pentadecapeptide
BPC 157, much like the one-time engraftment of neural
stem cells [16] or bone marrow stromal cells [17] into
the lesion site. This experiment will provide evidence
that BPC 157 treatment can recover tail function, resolve
spasticity, and improve neurologic recovery.
Materials and methods
Animals
Wistar albino male rats (aged 12 weeks, 350–400 g b.w.)
were bred in-house (the animal facility at the Department of Pharmacology, School of Medicine, Zagreb,
Croatia; registered by Directorate of Veterinary; Reg. No:
HR-POK-007), acclimated for 5 days, and randomly
assigned to experimental groups (at least 6 animals per
experimental group and interval). The experiments were
approved by the Local Ethics Committee. The laboratory
animals were housed in PC cages in conventional laboratory conditions at a temperature of 22.4 °C, a relative
humidity of 40–70%, and a noise level of 60 dB. Each
cage was identified by the date, study number, group,
dose, number, and sex of each animal. Fluorescent lighting provided illumination 12 h per day. A standard GLP
diet and fresh water were provided ad libitum. Furthermore, all experiments were carried out under a blind
protocol, and the effects were assessed by examiners
who were completely unaware of the protocol. We certify that government regulations concerning the ethical
use of animals were adhered to during this research.
Drugs
The pentadecapeptide BPC 157 (GEPPPGKPADDAGLV,
M.W. 1419) (Diagen, Ljubljana, Slovenia) dissolved in
0.9% NaCl was used in all experiments [1–11]. The
peptide BPC 157 is part of the sequence of the human
gastric juice protein BPC and is freely soluble in water
and 0.9% NaCl at pH 7.0. BPC 157 was prepared as
described previously with 99% high-pressure liquid chromatography (HPLC) purification, expressing 1-des-Gly
peptide as an impurity [1–11].
Surgery and spinal cord injury
Deeply anesthetized (3% isoflurane, ketamine 50 mg/kg
b.w.) rats were subjected to laminectomy at lumbar level
L2–L3, which corresponds to the sacrocaudal spinal
cord (S2-Co1) as described previously [42]. A neurosurgical piston with a graduated force of 60–66 g was
placed over the exposed dura and left for 60 s to induce
a compression injury. After the piston was removed, the
muscle and skin incisions were closed. A single injection
(0.9% NaCl 5 ml/kg b.w.; pentadecapeptide BPC 157
200 μg/kg b.w. or 2 μg/kg b.w.) was administered intraperitoneally 10 min postinjury. Thereafter, the animals
were returned to their cages in pairs, and food and water
were provided ad libitum. According to previously
assigned interval groups (7, 15, 30, 90, 180, and 360
days), the animals were sacrificed with an overdose of
3% isoflurane. To establish secondary spinal cord injury,
four animals were sacrificed 10 min after spinal injury
immediately prior to the administration of therapy. Four
animals were subjected only to laminectomy without
spinal cord injury and sacrificed after 360 days.
Clinical evaluation
Tail motor function was scored 8 h and 1, 4, 7, 15, 30,
90, 180, and 360 days after injury (0—autotomy; 1—
complete loss of tail function; 2—maximum elevation of
1/4 of the tail length; 3—maximum elevation of 1/2 of
the tail length; 4—maximum elevation of 3/4 of the tail
length; 5—normal function). At the same intervals, the
tails were observed for spasticity; after manual stimulation with the standardized stretch/rub maneuver, the
tails were scored according to the Bennett scale [42]: 0—
normal phenotype; 1—flaccid tail; 2—hypertonic flexor
muscle with coiled and stiff tail; 3—hyperreflexia, e.g.,
coiling flexor spasm and clonus in response to light
touch or stretch; and 4—hypertonic flexor and extensor
muscles, clonus and hyperreflexia, the latter including a
positive curling reaction.
Electrophysiology recordings
Before sacrifice, the animals from the 30-, 90-, 180-, and
360-day postspinal cord injury interval groups were
placed in a wooden box with their tails exposed. Three
pairs of monopolar needles were stabbed 3 mm deep
into the tail 10, 60, and 100 mm caudal to the tail base.
Using a TECA 15 electromyography apparatus with a
signal filter between 50 Hz and 5 kHz, voluntary muscle
activity was recorded from the most caudal pair of electrodes, and the average motor unit potential (MUP) was
recorded. Thereafter, the compound motor action potential (CMAP) was recorded from the same pair of electrodes after stimulating the first and second electrodes
(a repetition of 1 Hz and a stimulus duration of 0.05 ms).
The amplitude, polyphasic changes, and the proximal and distal CMAP latencies were recorded, and the nerve
conduction velocity was calculated according to previous
studies [41, 43].
Histology
A 10-mm long piece of the spinal column (the L2–L3 vertebral body) and the surrounding muscle were collected
from each sacrificed animal and fixed in 4% formaldehyde
in phosphate buffer (pH 7.4). Upon fixation, the spinal
cord was decalcinated, dehydrated in graded ethanol solutions, and embedded in paraffin. Serial 5-μm crosssections were deparaffinated in xylene, rehydrated in
graded ethanol solutions, and stained with hematoxylin/
eosin and toluidine blue (Kemica, Croatia). Part of the
spinal cord gray and white matter was used for analysis
under light microscopy (magnification × 300). According
to previous studies [13, 33], the intensity and distribution
of the following pathological spinal cord changes were
evaluated semiquantitatively (0—no changes; 1—small or
focal changes; 2—moderate changes; 3—numerous confluent changes): (a) the hemorrhagic zone, (b) edema, (c) the
loss of neurons in anterior horn and intermediate gray
matter, (d) vacuoles, and (e) the loss of lateral and posterior spinal column tracts.
For peripheral nerve analysis, a 5-mm-long piece of
tail 15 mm distal from the tail base was collected from
each sacrificed animal, fixed in 4% formaldehyde in
phosphate buffer (pH 7.4), decalcinated, and impregnated with 1% osmium tetroxide for a few days. The
specimens were dehydrated in graded ethanol solutions,
embedded in paraffin, cut into 5-μm sections, deparaffinated in xylene, rehydrated in graded ethanol solutions,
and mounted on glass slides. Representative field images
of four caudal nerves from each tail were taken using
light microscopy (magnification × 500) with a CCD camera using ISSA 3.1 software (VamStec, Zagreb) according
to a previous study [41]. Axonal myelination was analyzed according to the following quantifications: (a) the
total number of myelinated axons per 10,000 μm2
; (b)
the number of myelinated axons with a diameter ≥ 7 μm
per 10,000 μm2
; and (c) the average axonal diameter.
Statistical analyses
Scoring data are expressed as the median, min, and max
and were analyzed by Kruskal–Wallis ANOVA (P values
< 0.05 were considered significant) followed by the
Mann–Whitney U test (P values < 0.025 were considered
significant) with Bonferroni correction; these tests are
considered nonparametric alternatives to one-way
ANOVA and Student’s t test. Numeric data are
expressed as the mean ± standard deviation (SD) and
were analyzed by one-way ANOVA followed by LSD
test. The statistical program Statistica for Windows, ver.12.1 (StatSoft Inc. Tulsa, OK, USA) was used for statistical analysis. P values < 0.05 were considered significant.
Results
Clinical examinations
Tail motor function score
As expected, the tail motor function scores demonstrated persistent debilitation in the rats that underwent
spinal cord injury and received saline postinjury.
In contrast, after initial disability, the rats that underwent spinal cord injury and received BPC 157 exhibited
consistent improvement in motor function compared to
that in the corresponding controls. In particular,
from day 180, autotomy was noted in the rats that
underwent spinal cord injury but not in those that had
been treated with BPC 157.
Tail spasticity
Interestingly, the development of spasticity began
earlier in the rats that underwent spinal cord injury
and had been treated with BPC 157 than in the corresponding controls. However, the controls exhibited
sustained spasticity until the end of the experiment
(day 360) while the BPC 157 rats exhibited resolved
spasticity by day 15).
Histology results
Before the initiation of therapy, at 10 min after injury
induction, a large hemorrhagic zone was present over
the lateral and posterior white columns in all of the
rats, but there were no changes in the gray matter.
Notably, after the application of saline or BPC 157, the
injury progression in the rats from the different
experimental groups was fundamentally different.
Beginning on day 7, vacuoles and the loss of posterior and lateral spinal column tracts were observed
instead of hemorrhagic areas in all controls, disturbances that were largely counteracted in the BPC
157-treated rat. Likewise,
beginning on day 7, the controls exhibited edema
and the loss of neurons in the anterior horn and
intermediate gray matter, disturbances that were
largely counteracted the in BPC 157-treated rats.
While the significance of this finding remains to be
determined, it is probably worth mentioning that a
decrease in the number of large myelinated axons in rat
caudal nerves was observed in all animals until day 30,
with a markedly greater number in controls and fewer in
injured rats that received BPC 157 treatment. Interestingly, after 180 days, recovery occurred, and the number
of large myelinated axons in the controls reached that in
the BPC 157-treated rats, and this finding persisted
through the end of the experiment.
Electrophysiology results
Based on a well-known phenomenon in peripheral nerve
injury (i.e., as the number of preserved motoneurons
decreases, the MUP (giant potential) in the tail muscle
increases), it is conceivable that the BPC 157-treated rats
that underwent spinal cord injury and were subjected to
EMG recordings exhibited a markedly lower MUP in the
tail muscle than that in the corresponding controls. Consistently, the motor nerve conduction
study confirmed the absence of demyelinated processes in the tail caudal nerves after spinal cord injury (the
CMAP showed normal biphasic potentials, similar
amplitudes, and similar conduction velocities in all of
the rats) (Table 4).
Discussion
This study attempted to demonstrate that the application
of the stable gastric pentadecapeptide BPC 157 (by either
of the used regimens) can improve the symptoms of spinal
cord injury and lead to functional recovery in rats. In general, the one-time intraperitoneal application of the stable
gastric pentadecapeptide BPC 157 is much like the
engraftment of neural stem cells [16] or bone marrow
stromal cells [17] into the lesion site. One should consider
the primary phase lesion and hemorrhaging that results
from mechanical damage during SCI as well as the
secondary phase lesion that lasts several hours or even
several months and is accompanied by edema,
hemorrhage, inflammation, and cytotoxic edema [44–47]
and may extend to the white matter area and lead to white
matter degeneration and damage [48, 49]. This substantiates the evidence that the spared white matter holds the
key to the functional motor recovery of the hind limbs
after SCI and is closely correlated with the functional restoration of the paralyzed hind limbs [50–52]. On the other
hand, spontaneous and often substantial functional
improvements [53–55] after partial lesioning of the
spinal cord are associated with the spontaneous sprouting of axons in the corticospinal tract [56–58]
and the formation of neural circuits by spared spinal
cord tissue [26]; these processes lead to partial functional recovery [59] or the formation of the neural
fiber connection between the central pattern generator (CPG) and interneurons in the spinal cord,
which can enable rhythmic movement [60–62].
Thus, to illustrate these combining points (i.e., [13, 44,
63]), considering that white matter injury is the major
cause of functional loss after SCI [45, 52], it is important
to note that cysts and the loss of axons instead of
hemorrhagic areas were observed in the white matter in
all of the controls beginning on day 7 and that the rats
exhibited a tail motor score that persisted with only
small improvements, sustained debilitation, sustained
tail spasticity until the end of the experiment (day 360),
a decrease in the number of large myelinated axons in
the caudal nerve, a higher MUP (giant potential) in the
tail muscle, and a group of atrophic fibers that likely
represented a large unit that acquired many fibers
through collateral reinnervation and then degenerated.
Autotomy that occurs long after injury may appear as
pain that occurs below the level of the injury (belowlevel pain) [64, 65], and the late spontaneous worsening
may be the result of complete deafferentation of one or
several spinal segments the stimulation of the nerve
plexus, or dorsal root injury [66]. Together, these findings illustrate
definitive spinal cord injury with very
small spontaneous improvements in functional loss.
In contrast, it is possible that the administration of
BPC 157 counteracts these disturbances to lead to considerable functional recovery. The vacuoles and the loss
of axons in the white matter were largely counteracted
in BPC 157-treated rats (Table 1 and Fig. 3). This result
suggests that BPC 157-treated rats exhibit continual
improvement in motor function even before tissue recovery, as observed by microscopy assessment. The resolution of spasticity by day 15 (Fig. 2) suggests that BPC
157 administration prevents the chain of events after
spinal cord injury that is mediated by the loss of local
segmental inhibition and/or by an increased sensory afferent drive that results in the exacerbation of αmotoneuron activity [66]. These findings substantiate
the number of large myelinated axons in the caudal
nerve and the lower MUP in the tail muscle.
Likewise, autotomy was completely prevented, much
like in a previous study that showed recovery in BPC
157-treated rats that underwent traumatic nerve injury
[41]; this suggests the counteraction of the chain of
events that otherwise leads to painful sensations and refers to denervated regions and the preservation of one
or more spinal segments [41].
It is possible that BPC 157 may affect voltage-gated
sodium channels (VGSCs), which play a major role in
the generation and propagation of action potentials in
primary afferents [67].
The abnormal processing of sensory inputs in the CNS
[68]. Moreover, evidence that the compromised white
matter integrity of specific spinal pathways has been
linked to clinical disability [69–71], and cortical
reorganization [72] should be considered in relation to the
pleiotropic beneficial effect of BPC 157 administration observed in distinctive brain areas and lesions [32–40].
These beneficial effects include the counteractions of traumatic brain injury and severe encephalopathies after
NSAID overdose, insulin overdose, magnesium overdose,
and exposure to the neurotoxin cuprizone in a rat model
of multiple sclerosis [33–41]. These beneficial effects may
be due to the formation of detour circuits—which encompass spared tissue surrounding the lesion—and could reconnect locomotor circuits [69], thus enabling afferent
inputs to be processed and conveyed to the cortex [73]
and improving spinal reflexes, even below the injury [74].
Much like in the rats that underwent spinal cord
injury recovery, rats with other disorders that are treated
with BPC 157 maintain functional abilities that are
otherwise impaired; for example, consciousness is maintained after brain trauma, and BPC 157 counteracts
seizures, catalepsy akinesia, and severe muscle weakness
[33–41, 75, 76]. The effect of BPC 157 on muscle function is combined with the counteraction of increased
levels of pro-inflammatory and pro-cachectic cytokines
and of downstream pathways to abolish muscle cachexia
[2]. Likewise, BPC 157 ameliorates healing and recovers
the impaired function of severely injured muscles that
otherwise fail to spontaneously heal and plays a role
after complete transection, crush, and denervation injuries [77–80] and after succinylcholine intramuscular application, muscle lesion, neuromuscular junction failure,
fasciculations, paralysis, and hyperalgesia [81]. Likewise,
given that the gray matter is particularly vulnerable during the primary phase [44, 63], we should note that,
from day 7, the controls presented with edema and the
loss of motoneurons in the gray matter, disturbances
that were largely counteracted in BPC 157-treated rats
(Table 2 and Fig. 4).
In summary, this effect may be the cause or a consequence of the beneficial effects of BPC 157 on related
disturbances [1–11]. As demonstrated, BPC 157 counteracts free radical formation and free radical-induced lesions
[32, 82–84]. An interesting point would be the use of the
same dose range in BPC 157 studies [1–11]. Finally, further
studies should clarify the molecular pathways involved and
extend the one-time application (much like the engraftment
of neural stem cells [16] or bone marrow stromal cells [17]
into the lesion site) to the continuous application for the recovery of pre-existing spinal cord injury.
In conclusion, this manuscript tried to prove the therapeutic effects of BPC 157 in spinal cord injury using a rat
model. Spinal cord injury recovery was achieved in BPC
157-treated rats, meaning that this therapy affects the acute,
subacute, subchronic, and chronic stages of the secondary
injury phase. Thus, despite the limitations of rat studies,
the results showed that treatment with BPC 157 led to the
recovery of tail function and the resolution of spasticity and
improved the neurologic recovery; thus, BPC 157 may
represent a potential therapy for spinal cord injury.
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