Traumatic brain injury in mice and pentadecapeptide BPC 157 effect

Regulatory Peptides 160 (2010) 26–32

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Traumatic brain injury in mice and pentadecapeptide BPC 157 effect Mario Tudor, Ivan Jandric, Anton Marovic, Miroslav Gjurasin, Darko Perovic, Bozo Radic, Alenka Boban Blagaic, Danijela Kolenc, Luka Brcic, Kamelija Zarkovic, Sven Seiwerth, Predrag Sikiric ⁎ Department of Pharmacology, Medical Faculty University of Zagreb, Zagreb, Croatia Department of Pathology, Medical Faculty University of Zagreb, Zagreb, Croatia

a r t i c l e

i n f o

Article history: Received 25 April 2009 Received in revised form 11 November 2009 Accepted 12 November 2009 Available online 18 November 2009 Keywords: Traumatic brain injury Pentadecapeptide BPC 157 Mice

a b s t r a c t Gastric pentadecapeptide BPC 157 (GEPPPGKPADDAGLV, an anti-ulcer peptide, efficient in inflammatory bowel disease trials (PL 14736), no toxicity reported, improved muscle crush injury. After an induced traumatic brain injury (TBI) in mice by a falling weight, BPC 157 regimens (10.0 µg, 10.0 ng/kg i.p.) demonstrated a marked attenuation of damage with an improved early outcome and a minimal postponed mortality throughout a 24 h post-injury period. Ultimately, the traumatic lesions (subarachnoidal and intraventricular haemorrhage, brain laceration, haemorrhagic laceration) were less intense and consecutive brain edema had considerably improved. Given prophylactically (30 min before TBI) the improved conscious/unconscious/death ratio in TBI-mice was after force impulses of 0.068 N s, 0.093 N s, 0.113 N s, 0.130 N s, 0.145 N s, and 0.159 N s. Counteraction (with a reduction of unconsciousness, lower mortality) with both µg- and ng-regimens included the force impulses of 0.068–0.145 N s. A higher regimen presented effectiveness also against the maximal force impulse (0.159 N s). Furthermore, BPC 157 application immediately prior to injury was beneficial in mice subjected to force impulses of 0.093 N s-TBI. For a more severe force impulse (0.130 N s, 0.145 N s, or 0159 N s), the time-relation to improve the conscious/ unconscious/death ratio was: 5 min (0.130 N s-TBI), 20 min (0.145 N s-TBI) or 30 min (0.159 N s-TBI). © 2009 Elsevier B.V. All rights reserved.

1. Introduction Traumatic brain injury (TBI) is a major cause of death and disability worldwide, but lacks clinically proven effective management with neuroprotective agents to limit pathophysiologic cascades or enhance repair. The enormous burden of TBI, however, clearly supports the need for such neuroprotective agents. Several peptide growth factors were found to be effective [1,2]. Thereby, we focus on a so far not investigated peptide therapy for traumatic brain injury. We attempted to achieve a counteraction of these events by the stable gastric pentadecapeptide BPC 157 [3–40] (GEPPPGKPADDAGLV, MW 1419, a small anti-ulcer peptide protecting endothelium [3–14,16,17] that may also affect many central disturbances [16–22] and NO-system [4,13,15,19] and act as free radical scavenger [36], efficient in inflammatory bowel disease trials (PL 14736) [23,24] and various wound treatment [25–35], no toxicity reported [23,24]). Interestingly, it was an effective therapy of muscle crush injury in rats [34,35]. The significance and the possible role of this peptide stable in human gastric juice in the gastrointestinal tract were recently emphasized [37–40]. ⁎ Corresponding author. Department of Pharmacology Medical Faculty University of Zagreb, Salata 11, POB 916, 10000 Zagreb, Croatia. Tel.: +385 1 4566 833, +385 1 4566 834; fax: +385 1 4920 050. E-mail address: [email protected] (P. Sikiric). 0167-0115/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2009.11.012

Particularly, as an agent with neuroprotective properties [5], it both protected somatosensory neurons against capsaicin neurotoxicity and restored their function [5]. Likewise, pentadecapeptide BPC 157 particularly attenuated neurotoxin 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) damage, including mortality [16] (of note, neuroprotection by immunophillin ligands lack the effect on MPTPtoxicity 41), significantly affected the dopamine system [16–18] (a system also implicated in the traumatic brain injury course) [42] and prevented/reversed catalepsy or stereotypy due to central dopamine system failure induced by various procedures [16–18]. Accordingly, regional serotonin synthesis in the rat brain, assessed by α-methyl-Ltryptophan autoradiographic measurements showed that, BPC 157 given peripherally may readily cross the blood–brain barrier, affect region-specific brain 5-HT synthesis in rats leading to significantly increased synthesis in the substantia nigra (compacta and reticulata) structure and counteract serotonin syndrome [21,22]. Also, in addition to several other effects of BPC 157 (such as counteraction of acute and chronic gastric lesions, blood pressure modulation and prevention/reversal of chronic heart failure), BPC 157's antagonization of acute and chronic ethanol intoxication was found to be an effect that is at least partly NO-dependent [4,13,15,19]. Thereby, following TBI, we attempted to highlight the possible beneficial effects of pentadecapeptide BPC 157 in preventing immediate unconsciousness and death during the earliest post-injury period. Then, at 24 h after injury, in surviving mice subjected to TBI,

M. Tudor et al. / Regulatory Peptides 160 (2010) 26–32

we described the attenuating effect of BPC 157 on the intensity and distribution of traumatic lesions (subarachnoidal and intraventricular haemorrhage, brain laceration, haemorrhagic laceration) and consecutive brain edema. 2. Materials and methods

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2.3.4. Lower doses of pentadecapeptide BPC 157 The injury severity (ii) that could be produced by the falling weight (height 105 mm, kinetic energy 0.067 J and force impulse 0.093 N s) and the application time of 30 min before injury induction were selected to investigate the effects of lower doses of pentadecapeptide BPC 157, such as 100.0 pg, 10.0 pg and 1.0 pg/kg (while control-mice simultaneously received an equivolume of saline (5 ml/kg)).

2.1. Animals 2.4. Injury assessment Male, Albino NMRI (25–30 g) Hanover mice, were used in all of the experiments approved by the Local Ethic Committee, assessed by observers unaware about the given treatment. Animals were housed in air-conditioned rooms (T = 22+/−2 °C; relative humidity: 55– 65%) with a light–dark cycle of 12 h:12 h (light from 0700 to 1900 h) and food and water available ad lib. 2.2. Drugs Medication, without carrier or peptidase inhibitor, includes pentadecapeptide BPC 157 (a partial sequence of human gastric juice protein BPC, freely soluble in water at pH 7.0 and in saline; a peptide with 99% (HPLC) purity (1-des-Gly peptide as impurity), manufactured by Diagen, Ljubljana, Slovenia, GEPPPGKPADDAGLV, M.W. 1419 [3–9,11–22,25,28–36,38,40], dissolved in saline (10.0 µg, 10.0 ng, 100.0 pg, 10.0 pg and 1.0 pg/kg b.w.)), or saline (5.0 ml/kg), applied intraperitoneally. 2.3. Injury induction and medication application 2.3.1. Injury levels examined Various injury levels were examined by adjusting the height of the falling weight (diameter 18 mm, length 23 mm, weight 65 g), and kinetic and potential energy (Ek and Ep) (J) (Ek = Ep = m (weight (kg)) × g (gravity acceleration constant (9.8195 m/s2)) × h (height (m))) and force impulse pffiffiffi (N s) (F (force (N)) × t (time (s)) = m × v (velocity (m/s) = m × 2 × g × h). The severity of damage to the head of the mice were calculated as follows: (i) height 55, kinetic energy 0.035 J, force impulse 0.068 N s; (ii) height 105 mm, kinetic energy 0.067 J and force impulse 0.093 N s; (iii) height 155 mm, kinetic energy 0.099 J and force impulse 0.113 N s; (iv) height 205 mm, kinetic energy 0.131 J and force impulse 0.130 N s; (v) height 255 mm, kinetic energy 0.163 J and force impulse 0.145 N s; (vi) height 305 mm, kinetic energy 0.195 J and force impulse 0.159 N s. 2.3.2. Outcome of the maximal injury that could be improved To identify the outcome of the maximal injury that could be improved in TBI-mice by therapy application before injury, the intensity of the injury was gradually increased, beginning with injury i, ii, iii, iv, v and ending with the maximal injury (vi). Medication was given intraperitoneally at 30 min before injury induction. Pentadecapeptide BPC 157 was applied using previously effective regimens (10.0 µg, 10.0 ng/kg b.w.) [3–9,11–22,25,28–36,38,40]. The mice from the control group received at the time of medication an equivolume of saline (5 ml/kg i.p.). 2.3.3. Period before the onset of beneficial effect To determine the time that takes a protective effect, the shortest period elapsed after the medication application and the onset of its beneficial effects, the application times were: immediately before injury induction, 5 min or 10 min or 15 or 20 or 25 min or 30 min before injury induction. The injury severity that could be produced by the falling weight, was chosen as follows: (ii) height 105 mm, kinetic energy 0.067 J and force impulse 0.093 N s; (iv) height 205 mm, kinetic energy 0.131 J and force impulse 0.130 N s; (v) height 255 mm, kinetic energy 0.163 J and force impulse 0.145 N s; (vi) height 305 mm, kinetic energy 0.195 J and force impulse 0.159 N s.

The number of mice that remained conscious, or became unconscious or were killed by the head injury was calculated immediately after injury induction (force impulses of 0.068 N s, 0.093 N s, 0.113 N s, 0.130 N s, 0.145 N s, and 0.159 N s). The presence of the righting reflex was taken as a sign of consciousness while the absence of the righting reflex was taken as unconsciousness. Alternatively, in mice that initially survived TBI induced by a force impulse of 0.093 N s (injury severity (ii)), mortality was calculated for the 24 h post-injury period. At the end of the 24 h post-injury period, the TBI-mice were sacrificed (Table 1). The brains of the mice subjected to the TBI-force impulse of 0.093 N s (injury severity (ii)) that i.p. received BPC 157 (10.0 µg, 10.0 ng/kg) or saline, at 30 min before injury or immediately before injury, sacrificed either immediately after injury or at 24 h after injury, were fixed in 10 % formalin during 2 days. Upon fixation, the brains were grossly inspected and cut by consecutive coronal sections. Brain slabs were dehydrated in graded ethanol and embedded in paraffin. Paraffin blocks were cut into 5 µm thin slices. Paraffin slices were deparaffinated in xylene, rehydrated in graded ethanol and stained with haematoxylin and eosine (Kemika, Croatia). Two registered pathologists (K.Ž. and S.S.) diagnosed each specimen independently. Intensity and distribution of traumatic lesions (subarachnoidal and intraventricular haemorrhage, brain laceration, haemorrhagic laceration) and consecutive brain edema were described and evaluated semiquantitatively (0–3). While 0 generally indicated no changes, lesions were subsequently scored as follows: brain edema: 1 — weak diffuse and/or perifocal; 2 — moderate; 3 — strong and generalized; traumatic non-haemorrhagic laceration: 1 — small or focal; 2 — big or numerous confluent; traumatic haemorrhagic laceration: 1 — small or focal; 2 — big or numerous confluent; subarachnoidal haemorrhage: 1 — focal; 2 — generalized; intraventricular haemorrhage: 1 — thin bleeding; 2 — extensive bleeding (Table 2). 2.5. Statistical analysis Statistical analysis was performed using the program GraphPad Prism 5.00 for Windows. 2-sided Fisher exact test was used to compare the proportions between the groups. To analyze differences between categorical group data Kruskal–Wallis and post hoc Mann Whitney U test were used. Statistically significant difference was considered at P b 0.05.

Table 1 Severity of damage (i–vi) to the head of the mice: height, mm, kinetic energy, J, force impulse, Ns. Severity of damage (i–vi) to the head of the mice

Height, mm

Kinetic energy, J

Force impulse, Ns

i ii iii iv v vi

55 105 155 205 255 305

0.035 0.067 0.099 0.131 0.163 0.195

0.068 0.093 0.113 0.130 0.145 0.159

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Table 2 Intensity and distribution of traumatic lesions and consecutive brain edema. Semiquantitative scoring (0–3)

Intensity and distribution of traumatic lesions and consecutive brain edema evaluated semiquantitatively (0–3) Brain edema

Traumatic non-haemorrhagic laceration

Traumatic haemorrhagic laceration

Subarachnoidal haemorrhage

Intraventricular haemorrhage

0 1 2 3

No changes Weak diffuse and/or perifocal Moderate Strong and generalized

No changes Small or focal Big or numerous confluent

No changes Small or focal Big or numerous confluent

No changes Focal Generalized

No changes Thin bleeding Extensive bleeding

3. Results 3.1. Immediate assessment Commonly, the applied TBI procedures (force impulses of 0.068 N s, 0.093 N s, 0.113 N s, 0.130 N s, 0.145 N s, and 0.159 N s, injury severity i–vi) produced unconsciousness as well as fatal outcomes. Elevation of height was in correlation with the gradually increased severity of produced TBI (Table 3). In general, presenting as the improved conscious/unconscious/death ratio in TBI-mice, the pentadecapeptide BPC 157 regimens resulted with marked attenuation and improved early outcome. The improved conscious/unconscious/death ratio in TBI-mice that received BPC 157 was commonly seen after TBI was produced with force impulses of 0.068 N s, 0.093 N s, 0.113 N s, 0.130 N s, 0.145 N s, and 0.159 N s, i–vi. The counteracting effect (less unconsciousness as well as fatal outcomes) was commonly seen with µg and ng (i–v, force impulse 0.068–0.145 N s) up to the maximal TBI severity (vi; force impulse 0.159 N s) at which effectiveness was shown only with the µg-regimen (Table 1). These beneficial effects with the conscious/unconscious/death ratio in TBI-mice could be not achieved after the application of lower dose regimens given at 30 min before injury induction (ii, force impulse 0.093 N s) (i.e., 24/6/0 (100 pg), 16/14/0(10 pg), 18/12/0 (1 pg), P N 0.05 vs. control). Assuming the importance of the time-relation with respect to the delivery of the beneficial effect to the injured brain (i.e., counteraction of the less severe TBI produced by lower force impulse would require shorter periods while more severe TBI produced by a higher force impulse would require more time), we showed an interesting timeshift of initiation of BPC 157 beneficial effects. Even an application immediately before injury induction was beneficial in mice subjected to a force impulse of 0.093 N s-TBI (ii). For stronger TBI (iv, v, vi; force impulses of 0.130 N s, 0.145 N s, and 0159 N s), the time-relation to improve conscious/unconscious/death ratio was: 5 min (iv; 0.130 N sTBI), 20 min (v; 0.145 N s-TBI) or 30 min (0.159 N s-TBI) (Table 4). 3.2. Postponed assessment Without therapy, after TBI using a force impulse of 0.093 N s (ii), although initially without immediate mortality, an increasing post-

poned mortality characterized the subsequent post-injury period up to 24 h (Table 3, Fig. 1). Commonly, at 24 h after injury, the surviving animals exhibited lacerations of brain tissue without haemorrhage in the fronto-parietal cortex and subcortical white matter beneath the impact (coup lesion). Also usually, animals had lacerations with haemorrhage in the same regions. No inflammatory response was noted in both lesions. Regularly, intraventricular acute bleeding was without inflammatory response. Subarachnoidal bleeding was regularly diffuse, occasionally located beneath the impact in the fronto-parietal region. No inflammatory response was noted. Interstitial strong and diffuse edema (predominantly perifocal and in white matter) was commonly noted. Unlike TBI-controls, the post-injury mortality was minimal in BPC 157 regimens (Table 5, Figs. 1 and 2). This was in concordance with no influence on non-haemorrhagic brain lesions. The brain edema, number and size of haemorrhagic traumatic lacerations were attenuated in mice that had been injected with BPC 157 at 30 min before injury and in those that had been treated immediately before injury. The intensity of subarachnoidal bleeding was attenuated when BPC 157 was given immediately before injury, while the intraventricular haemorrhage was attenuated when BPC 157 was given at 30 min before injury. 4. Discussion We reported a consistent (clinical and microscopical) beneficial effect of BPC 157 in mice after traumatic brain injury with an escalating severity, as noted in Section 2. This was backed by the consistent injury course (i.e., instant unconsciousness (absence of the righting reflex) and death, postponed deleterious outcome presented by prominent brain edema, numerous, variably sized haemorrhagic traumatic lacerations, subarachnoidal and intraventricular haemorrhage frequently resulting with fatality throughout a 24 h post-injury period) and investigations carried out using increasing force impulses to produce TBI. In accordance with the primary injury to the brain that initiates a secondary injury process of TBI, the BPC 157 counteracting effects concerned the immediate consequences of severe head injury in mice, the preserved consciousness (with less unconsciousness and fatality) as well as

Table 3 TBI severity and the beneficial effects of BPC 157 application (10.0 μg, 10 ng/kg i.p.) on mice with various TBIs induced. Time (min) elapsed between medication application and TBI induction was 30 min. P b 0.05 vs. control indicates the TBIs severity and the application of BPC 157 regimens when beneficial effect was obtained. TBI severity (i–vi) Force impulse

(i): 0.068 N s (ii): 0.093 N s (iii): 0.113 N s (iv): 0.130 N s (v): 0.145 N s (vi): 0.159 N s

Medication given at 30 min before injury induction Saline 5 ml/kg i.p.

BPC 157 10.0 µg/kg i.p.

Number of mice that was

Number of mice that was

BPC 157 10.0 ng/kg i.p. Number of mice that was

Conscious

Unconscious

Killed

Conscious

Unconscious

Killed

Conscious

Unconscious

Killed

21 17 12 9 1 0

9 13 18 18 21 6

0 0 0 3 8 24

30 28 27 24 12 3

0 2 3 6 15 17

0 0 0 0 3 10

29 27 21 22 9 1

1 3 9 6 15 6

0 0 0 2 6 23

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Fig. 1. Characteristic presentation of the traumatic brain injury in mice at 24 h postinjury without therapy. A. Numerous lacerations without haemorrhage in frontoparietal cortex and subcortical white matter beneath the impact area in control animal. (HE, 40×). B. Subarachnoidal bleeding beneath the impact in fronto-parietal region of control animal. (HE, 40×). C. Haemorrhagic laceration and diffuse edema in basal area (contra coup lesion) in control animal. (HE, 200).

attenuated and improved the postponed deleterious outcome (lower brain edema, significantly lower number and size of haemorrhagic traumatic lacerations, significantly lower intensity of subarachnoidal bleeding, significantly less intraventricular haemorrhage shown microscopically, declined fatality) (Tables 3, 4 and 5, Figs. 1 and 2). To properly evaluate the achievement of the used BPC 157-therapy it is important to know that no carrier was used [3–36], and thereby, the obtained beneficial effect can be directly attributed to this peptide (unlike other peptides that use different carriers, and peptide + carrier(s)-complex which bear considerable methodological/activity dilemmas)) [43–45]. What's more, the accuracy of the alpha-[14C] Fig. 2. Characteristic presentation of the traumatic brain injury in mice at 24 h postinjury with BPC 157 therapy. A. Traumatic laceration without haemorrhage in corticosubcortical fronto-parietal region beneath the impact in animals pretreated with BPC 157. (HE, 40×). B. Third ventricle without bleeding in animals pretreated with BPC 157. (HE, 40×). C. Small non-haemorrhagic lacerations and average edema in corticosubcortical fronto-parietal region beneath the impact in animals pretreated with BPC 157. (HE, 40×). D. Small non-haemorrhagic lacerations and average edema in corticosubcortical fronto-parietal region beneath the impact in animal cotreated with BPC. (HE, 100×). E. Non-haemorrhagic lacerations and average edema in periventricular area in animals cotreated with BPC 157. (HE, 100×).

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methyl-L-tryptophan (alpha-MTrp) autoradiographic method, supports the premise that given peripherally, BPC 157 may have regionspecific influences on the brain (i.e., given peripherally, acutely or chronically, BPC 157 specifically influenced brain 5-HT synthesis in rats (i.e., dorsal thalamus, hippocampus, lateral geniculate body, hypothalamus, dorsal raphe nucleus, substantia nigra, medial anterior olfactory nucleus, lateral caudate, accumbens nucleus, superior olive)) [21]. Also, the safe application of pentadecapeptide BPC 157 (LD1 not achieved, limit test negative, no side effects in patients) [23,24] along with the beneficial effect obtained in the traumatic brain injury, most likely suggests that it may be different from systemic administration of numerous factors (e.g., NGF) that have also by the way, been found to display severe side effects independent of the nerve injury [46]. However, the precise mechanism(s) of this beneficial effect of pentadecapeptide BPC 157 remain elusive. Even so, the salvage of traumatic brain injury (i.e., reduction of immediate mortality and subsequent complication rate) and an efficient healing in BPC 157 mice were repeatedly obtained. The beneficial effect was affected by the dosage, the timing of administration and severity of the applied injury. Together, these findings probably indicate several possibilities. The effect on primary injury may be interesting, since primary injury is associated with the initial mechanical insult, resulting in immediate and often irreversible damage to neuronal cell bodies, dendrites, axons, glial cells, and brain vasculature. Generally, the primary injury also results in tissue deformation and compression, leading to seizures, respiratory depression, apnoea, ischemic, and hypoxic damage, resulting in cellular injury [47,48]. On the other hand, the secondary injury is caused by an incompletely understood and complex cascade of physiological and biochemical factors continuing for hours to days post-injury with resulting progressive tissue damage [47–50]. In this respect, presenting the beneficial effect on TBI induced by the force impulse of 0.093 N s-TBI (ii) obtained with BPC 157 application at 30 min before injury or immediately before injury, it may be particularly interesting that after 24 h we demonstrated no influence of BPC 157 on non-haemorrhagic brain lesions. The brain edema, number and size of haemorrhagic traumatic lacerations were

attenuated in mice that had been injected with BPC 157 at 30 min before injury and in those that had been treated immediately before injury. The intensity of subarachnoidal haemorrhage was attenuated when BPC 157 was given immediately before injury. The intraventricular haemorrhage was attenuated when BPC 157 was given at 30 min before injury. In addition, attenuation of the brain edema was consistently more expressed in BPC 157 pretreatment. Together, these findings may suggest that BPC 157 may modify the primary injury to the brain and the immediate events that initiate a secondary injury process. Additionally, the time-relation presented with BPC 157 application an immediate effect on TBI up to the force impulse of 0.093 N s-TBI (ii) while by the time the higher TBI and the higher force impulses could be also opposed (force impulses of 0.130 N s, 0.145 N s, and 0.159 N s, iv–vi). It seems that BPC 157 processes extend depending on its time of application, increasing as time elapses (where even more severe lesions could be additionally counteracted). Possibly, this time lag, in addition to a direct effect, reflects an increasing chain of triggered events involved after application of the peptide being activated to adapt the brain to subsequent major injuries. For instance, we repeatedly showed that BPC 157 (µg–ng/kg dosage range) can greatly overwhelm the effect of corticosteroids (mg/kg) [12,14,33,35] (i.e., the assumption had been made that the beneficial effects of steroids in treating head injuries were related to the reduction of edema) [51]. Thereby, the beneficial effects of BPC 157 may be generally, within the described traumatic brain injury, acute blood–brain barrier (BBB) opening, entry of leukocytes into the injured brain [52,53], release of free oxygen radicals causing cellular damage, release of inflammatory cytokines and particularly, BPC 157 may have an effect through NO, a molecule that acts as a neuronal messenger, immunomodulator, and vasoactive substance, which may play an important role in the posttraumatic sequelae [54] that have been implicated in posttraumatic neuropathologic damage. To this point, the effect of BPC 157 in the substantia nigra (compacta and reticulata) structure may be indicative. Namely, it increased 5-HT synthesis (acutely and chronically) [21,22], attenuated motor abnormalities (tremor, akinesia, catalepsy) [16–18], abolished

Table 4 Time-relation between the medication time and the onset of the beneficial effects of BPC 157 application (10.0 μg, 10 ng/kg i.p.) on mice with various TBIs induced. Time (min) elapsed between medication application and TBI induction using escalating force impulse application. P b 0.05 vs. control indicates the time points of the application of BPC 157 regimens when significant beneficial effect was obtained. Time (min) elapsed between medication application and TBI induction

0

5

10

15

20

25

Medication given at various time points before injury induction Saline 5 ml/kg i.p.

BPC 157 10.0 µg/kg i.p.

BPC 157 10.0 ng/kg i.p.

Number of mice that was

Number of mice that was

Number of mice that was

Conscious/Unconscious/Killed

Conscious/Unconscious/Killed

Conscious/Unconscious/Killed

TBI severity (ii,iv–vi) Force impulse

TBI severity (ii,iv–vi) Force impulse

TBI severity (ii,iv–vi) Force impulse

(ii): 0.093 N s

(iv): 0.130 N s

(v): 0.145 N s

(vi): 0.159 N s

(ii): 0.093 N s

(iv): 0.130 N s

(v): 0.145 N s

(vi): 0.159 N s

(ii): 0.093 N s

(iv): 0.130 N s

(v): 0.145 N s

(vi): 0.159 N s

17 13 0 17 13 0 17 13 0 17 13 0 17 13 0 17 13 0

9 18 3 9 18 3 9 18 3 9 18 3 9 18 3 9 18 3

1 21 8 1 21 8 1 21 8 1 21 8 1 21 8 1 21 8

0 6 24 0 6 24 0 6 24 0 6 24 0 6 24 0 6 24

28 2 0 28 2 0 28 2 0 28 2 0 28 2 0 28 2 0

20 10 0 24 6 0 24 6 0 24 6 0 24 6 0 24 6 0

7 20 3 7 20 3 7 20 3 7 20 3 12 15 3 12 15 3

1 6 23 1 6 23 1 6 23 1 6 23 1 6 23 1 6 23

28 2 0 28 2 0 28 2 0 28 2 0 28 2 0 28 2 0

20 10 0 20 10 0 24 6 0 24 6 0 24 6 0 24 6 0

7 20 3 7 20 3 7 20 3 7 20 3 7 20 3 12 15 3

1 6 23 1 6 23 1 6 23 1 6 23 1 6 23 1 6 23

M. Tudor et al. / Regulatory Peptides 160 (2010) 26–32

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Table 5 Intensity of traumatic lesions (subarachnoidal haemorrhage, IVH, brain laceration, haemorrhagic laceration and consecutive brain edema) presenting in mice at 24 h post-injury that survived initial TBI induction using a force impulse of 0.093 N s and that had been initially treated with BPC 157 application (10.0 μg, 10 ng/kg i.p.) either immediately before injury or at 30 min before injury induction. P b 0.05 vs. control. Application time

Medication

Mortality rate % through 24 h post-injury period

Intensity of traumatic lesions (subarachnoidal haemorrhage, intraventricular haemorrhage, brain laceration, haemorrhagic laceration, non-haemorrhagic l aceration) and consecutive brain edema presenting in mice at 24 h post-injury min/med/max

Number of mice Total number Edema Non-haemorrhagic Haemorrhagic traumatic Subarachnoidal Intraventricular that died of mice traumatic lacerations lacerations haemorrhage haemorrhage 30 min before injury

Saline 5 ml/kg i.p. 15 BPC 157 10 µg/kg i.p. 1 BPC 157 10 ng/kg i.p. 2 Immediately before injury Saline 5 ml/kg i.p. 16 BPC 157 10 µg/kg i.p. 5 BPC 157 10 ng/kg i.p. 7

30 30 30 30 30 30

1/3/3 0/1/2 1/1/2 2/3/3 0/1/3 1/1/3

lethality caused by application of MPTP which is a Parkinsongenic neurotoxin, and attenuated the free radical dopamine cellular damage in the substantia nigra [16], thereby presenting that it might also attenuate other free radical injuries [36]. It might also counteract the release of free oxygen radicals which cause cellular damage in TBImice. Pentadecapeptide BPC 157 also reduced the levels of LTB4, TXB2 and MPO in both serum and inflamed tissues [10,29]. Its beneficial effect (i.e., mucosa and wound healing, particularly in crushed muscle healing) parallels the reduction of inflammatory cells, edema and haematoma formation [12–14,25–35]. It may be of importance to mention that pentadecapeptide BPC 157 interacted with nitric oxide (NO) and NO-agents, reduced big endothelin-1(BET-1) serum values [4,13,15,19], maintained endothelium integrity [3–14,16,17] and showed a marked angiogenic effect [11–14,25–35], presenting the role of NO (the cerebral endothelium as the source of protective NO to restore arterial reactivity following CCI brain injury) [54] and the endothelin vasoconstrictive peptide in the deterioration of cerebral perfusion [55]. Finally, these therapeutic effects of BPC 157 could be the consequence of its suggested significance in the gastrointestinal tract [38–40] presenting that the peptide may also act indirectly from a binding site in the gut at some visceral receptive relay of the central nervous system [56]. In conclusion, these results should be viewed with numerous compounds and neuroprotective strategies more extensively discussed, evaluated and reviewed elsewhere [57,58]. However, brain trauma results in brain damage and dysfunction from both primary injury (due to biomechanical effects) and subsequent secondary damage due to activation of pathophysiologic cascades [59], and we evidenced the preserved consciousness and reduced mortality immediately after trauma in pentadecapeptide BPC 157-mice and subsequently, markedly reduced mortality, lowered brain edema, lowered the number and size of haemorrhagic traumatic lacerations, and lowered the intensity of subarachnoidal bleeding with significantly less intraventricular haemorrhage. On the other hand, after contusion, BPC 157 (given locally or intraperitoneally) improved crushed muscle healing, macroscopically and microscopically showed less post-injury haematoma and edema. Functionally, there was a complete restitution of function [34,35]. References [1] Saatman KE, Contreras PC, Smith DH, Raghupathi R, McDermott KL, Fernandez SC, Sanderson KL, Voddi M, McIntosh TK. Insulin-like growth factor-1 (IGF-1) improves both neurological motor and cognitive outcome following experimental brain injury. Exp Neurol 1997;147(2):418–27. [2] Ray SK, Dixon CE, Banik NL. Molecular mechanisms in the pathogenesis of traumatic brain injury. Histol Histopathol 2002;17(4):1137–52 Oct. [3] Sikiric P, Seiwerth S, Grabarevic Z, et al. The beneficial effect of BPC 157, a 15 aminoacid peptide BPC fragment, on gastric and duodenal lesion induced by restraint stress, cysteamine and 96% ethanol in rats. A comparative study with H2 receptor antagonists, dopamine promoters and gut peptides. Life Sci 1994;54:PL63–8.

1/2/2 1/2/2 1/2/2 1/2/2 1/2/2 1/2/2

1/2/2 0/1/2 0/1/2 1/2/2 0/0/1 0/0/1

1/2/2 1/2/2 1/2/2 1/2/2 1/1/2 1/1/2

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