Peptide therapy with pentadecapeptide BPC 157 in traumatic nerve injury

Regulatory Peptides 160 (2010) 33–41

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Peptide therapy with pentadecapeptide BPC 157 in traumatic nerve injury Miroslav Gjurasin a, Pavle Miklic b, Bozidar Zupancic a, Darko Perovic a, Kamelija Zarkovic c, Luka Brcic c, Danijela Kolenc c, Bozo Radic a, Sven Seiwerth c, Predrag Sikiric a,⁎ a b c

Department of Pharmacology, Medical Faculty, University of Zagreb Medical School, Salata 11, POB 916, 10000 Zagreb, Croatia Department of Neurosurgery, University Hospital Center Zagreb, Zagreb, Croatia Institute of Pathology, Medical Faculty, University of Zagreb, Zagreb, Croatia

a r t i c l e

i n f o

Article history: Received 4 May 2009 Received in revised form 23 September 2009 Accepted 1 November 2009 Available online 10 November 2009 Keywords: Pentadecapeptide BPC 157 Rat Transected nerve

a b s t r a c t We focused on the healing of rat transected sciatic nerve and improvement made by stable gastric pentadecapeptide BPC 157 (10 µg, 10 ng/kg) applied shortly after injury (i) intraperitoneally/intragastrically/locally, at the site of anastomosis, or after (ii) non-anastomozed nerve tubing (7 mm nerve segment resected) directly into the tube. Improvement was shown clinically (autotomy), microscopically/ morphometrically and functionally (EMG, one or two months post-injury, walking recovery (sciatic functional index (SFI)) at weekly intervals). BPC 157-rats exhibited faster axonal regeneration: histomorphometrically (improved presentation of neural fascicles, homogeneous regeneration pattern, increased density and size of regenerative fibers, existence of epineural and perineural regeneration, uniform target orientation of regenerative fibers, and higher proportion of neural vs. connective tissue, all fascicles in each nerve showed increased diameter of myelinated fibers, thickness of myelin sheet, number of myelinated fibers per area and myelinated fibers as a percentage of the nerve transected area and the increased blood vessels presentation), electrophysiologically (increased motor action potentials), functionally (improved SFI), the autotomy absent. Thus, BPC 157 markedly improved rat sciatic nerve healing. © 2009 Elsevier B.V. All rights reserved.

1. Introduction We focused on the improvement of the healing after traumatic nerve injury [1–12], transected rat sciatic nerve injury healing and a peptide therapy, using a small, orally active, anti-ulcer peptide [13–22] — stable gastric pentadecapeptide BPC 157 (MW 1419) effective in trials for inflammatory bowel disease therapy [19–22]. Knowing also BPC 157's wound healing capability, increased collagen and new blood vessels formation, decreased myeloperoxidase (MPO) activity and inflammatory cell influx, including healing of transected muscle and tendon [13,14,23–32], we thought that it could also influence the healing of transected nerve injuries, although generalization is not always applicable, since for example, tacrolimus (FK506) does not have a consistent effect on wound healing [33]. Important fact is that BPC 157 has undoubtedly a positive effect on muscle healing [27–30], providing the importance of the suggested regeneration of the damaged intramuscular nerve branches [34]. BPC 157 shares some characteristics with neuroimmunophilin ligands, of which tacrolimus (FK506) has emerged as a particularly promising therapeutic agent important in a nerve injury [1,2]. Neuroimmunophilin ligands cross the blood–brain barrier and are orally effective in a variety of animal models of ischemia, traumatic

⁎ Corresponding author. Tel.: +385 1 4566 833; 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.2009.11.005

nerve injury and human neurodegenerative disorders [1]. BPC 157 given peripherally also crosses the blood–brain barrier influencing region-specific serotonin synthesis in the rat brain [35,36]. Unlike neuroimmunophilin ligands [3], it also exhibits a neurotropic effect attenuating 1-methyl-4-phenyl 1,2,3,6, tetrahydropyridine (MPTP) brain lesion and mortality [15]. Therefore, after sciatic nerve transection, BPC 157 was applied intraperitoneally, intragastrically or locally, at the site of anastomosis immediately after nerve anastomosis creation. In addition, after the nerve segment was dissected, BPC 157 was applied directly into the tube to completely fulfill the gap between the nerve stumps.

2. Materials and methods All experimental procedures were approved by the local Ethics Committee and assessed by the observers blinded about the treatment given. We used male Wistar Albino rats, 200 g body weight, randomly assigned, at least 10 rats per experimental group per period. The surgical procedures were performed under a dissection microscope (Opton, Oberkochen, Germany) in deeply anaesthetized rats (pentobarbital, 65 mg/kg i.p.). The right sciatic nerve was exposed and completely transected with microscissors, at 5 mm distal from foramen infrapiriforme, and the proximal and distal stumps anastomozed with three 10-0 monofilament epineurial sutures at each end (Ethilon, Ethicon Inc, USA). Alternatively, the

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M. Gjurasin et al. / Regulatory Peptides 160 (2010) 33–41

7 mm long nerve segment was dissected, and using two 10-0 monofilament epineurial sutures at each end, the severed nerve ends were sutured to 1 mm inside a 9-mm silicone tube (Heyer Schulte, USA; internal diameter 1.20 mm), forming a 7 mm nerve gap with a volume of 17.7 µl. After the operation, all muscle incisions and skin incisions were sutured (4-0 Vicryl (polyglactin 910, Ethicon, USA), Dermalon 2-0 (Davis & Geck, GB)). 2.1. Therapy Medication includes pentadecapeptide BPC 157 (a partial of sequence of human gastric juice protein BPC, freely soluble in water

at pH 7.0 and in saline); peptide with 99% (HPLC) purity (1-des-Gly peptide as impurity, manufactured by Diagen, Ljubljana, Slovenia, GEPPPGKPADDAGLV, M.W. 1419) [14–19,23–25,27–30,35–40]. BPC 157 dissolved in saline (10 µg/kg, 10 ng/kg) or saline (5.0 ml/kg) were applied intraperitoneally, intragastrically or locally, at the site of anastomosis (1 ml/bath) immediately after nerve anastomosis creation. After the tubing operation, to completely fulfill the gap with the tested solutions, an insulin needle with a volume of 17.7 µl of the pentadecapeptide BPC 157 (58 µg/ml, 58 ng/ml) or an equivolume of the saline was applied directly into the distal part of the tube, and the corresponding volume of the air was evacuated proximally with the other needle. Assessment procedure was carried out one or two

Table 1 Morphometrical and EMG analyses of sciatic nerve recovery after transection and nerve anastomosis, and therapy with stable gastric pentadecapeptide BPC 157 application. Mann– Whitney U-test ⁎p b 0.05 at least vs. control. Application immediately after nerve anastomosis creation Intra-peritoneal

Medication

Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg Saline Locally, at the site of anastomosis 1 ml/rat 5 ml/kg (1 ml/bath) BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg Intra-gastrical Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 0 µg/kg BPC 157 2 ng/rat 10 ng/kg Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg

Time after Conduction nerve injury velocity m/s

The diameter Diameter of of the axons myelinated fibers (μm) (μm)

Number of Thickness of Number of % of myelinated fibers myelin sheet myelinated area as % of neural tissue blood vessels/ 2 2 envelope(μm) fibers × 10 /mm on nerve cross section visual field

Min/Med/Max

Min/Med/Max Min/Med/Max Min/Med/Max Min/Med/Max

Min/Med/Max

Min/Med/Max

1 month

5.7/24.5/38.1

1.0/2.1/5.5

1 month

1.0/2.0/5.5

0.1/0.2/0.6

50/90/130

3.1/5.0/10.0

1.5/2.2/2.7

29.9/41.4/44.4⁎ 1.8/4.2/8.3⁎

1.5/4.6/8.7⁎

0.2/0.4/1.1⁎

200/250/270⁎

25.1/30.2/41.1⁎

2.0/2.9/3.8⁎

1 month

26.6/38.3/45.6⁎ 2.0/3.5/8.2⁎

2.0/4.4/8.5⁎

0.1/0.4/1.1⁎

200/240/260⁎

20.1/29.3/37.3⁎

1.7/2.7/3.1⁎

2 months

15.4/29.3/36.6

0.5/3.0/6.2

0.1/0.3/1.2

100/150/170

6.0/8.2/15.1

3.0/3.6/4.3

2 months

25.0/53.3/66.6⁎ 1.0/4.8/11.1⁎ 1.5/4.9/9.2⁎

0.3/0.7/1.5⁎

250/310/350⁎

44.2/59.0/61.3⁎

6.6/7.3/7.6⁎

2 months

30.5/39.3/46.6⁎ 1.0/4.9/10.7⁎ 1.5/4.8/8.5⁎

0.2/0.7/1.3⁎

240/290/340⁎

39.9/51.6/57.4⁎

5.9/6.6/6.9⁎

1 month

7.2/27.6/33.5

1.1/2.0/5.1

0.2/0.3/0.6

50/90/140

3.3/5.7/9.7

1.6/2.2/2.8

1 month

29.3/38.3/48.1⁎ 2.1/3.5/8.4⁎

2.5/3.5/8.4⁎

0.3/0.5/1.1⁎

190/230/240⁎

12.1/28.2/41.3⁎

2.2/3.0/3.7⁎

1 month

20.7/36.9/42.4⁎ 2.0/3.5/8.1⁎

2.4/3.4/8.0⁎

0.2/0.5/1.1⁎

160/250/250⁎

9.8/25.9/39.9⁎

1.9/2.5/2.9⁎

2 months

17.5/30.3/33.3

1.0/2.5/8.0

1.0/2.5/6.8

0.1/0.3/0.9

70/160/180

5.2/9.4/17.1

3.2/3.9/4.7

2 months

25/33.4/55.5⁎

2.1/5.4/10.3⁎ 2.5/5.5/10.1⁎ 0.3/0.7/1.4⁎

260/330/360⁎

45.0/59.4/62.5⁎

6.5/7.5/7.9⁎

2 months

22.4/37.8/43.9⁎ 2.1/4.9/9.9⁎

2.0/4.5/9.9⁎

0.4/0.8/1.2⁎

210/300/330⁎

42.7/57.8/60.6⁎

6.1/6.7/7.0⁎

1 month

1.9/26.6/40.1

1.0/2.1/5.0

0.1/0.2/0.6

40/100/130

4.0/6.1/11.0

1.4/2.0/2.4

1 month

32.2/44.0/65.2⁎ 1.1/3.5/9.5⁎

1.6/4.9/9.9⁎

0.2/0.4/1.2⁎

210/260/300⁎

22.3/30.7/43.4⁎

1.9/2.7/3.7⁎

1 month

28.3/40.4/48.2⁎ 1.1/3.6/10.0⁎ 1.7/3.9/9.1⁎

0.2/0.4/1.2⁎

230/250/310⁎

19.6/29.0/38.7⁎

1.5/2.4/2.9⁎

2 months

23.3/26.6/33.3

0.1/0.2/0.7

60/150/160

5.3/10.1/20.2

3.1/3.8/4.5

2 months

20.0/51.9/70.1⁎ 2.1/5.4/10.1⁎ 2.4/4.9/9.9⁎

0.2/0.6/1.3⁎

250/340/350⁎

40.3/53.6/60.6⁎

6.4/7.4/7.7⁎

2 months

30.3/41.3/49.8⁎ 2.1/4.9/9.9⁎

0.2/0.6/1.1⁎

250/300/320⁎

32.5/50.9/59.6⁎

5.8/6.8/6.9⁎

0.5/2.7/8.1

1.0/2.2/8.5

1.1/2.3/5.5

1.1/2.0/8.1

1.0/2.2/6.7

2.3/4.8/9.7⁎

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35

Table 2 Morphometrical and EMG analyses of sciatic nerve recovery after transection and the 7 mm long nerve segment was dissected, the severed nerve ends were sutured to 1 mm inside a 9-mm silicone tube, and therapy with stable gastric pentadecapeptide BPC 157 application. Mann–Whitney U-test p b 0.05 at least vs. control. Thickness of myelin sheet envelope (μm)

The diameter Diameter of of the axons myelinated fibers (μm) (μm)

Number of myelinated fibers × 102/ mm2

Number of blood vessels/ visual field

Application after tubing operation

Medication

Time after Conduction velocity m/s nerve injury Min/Med/Max

Min/Med/Max Min/Med/Max

Min/Med/Max

Min/Med/Max

Min/Med/Max

Min/Med/Max

An insulin needle with a volume of 17.7 µl of the pentadecapeptide BPC 157 58 µg, 58 ng/ml or an equivolume of the saline was applied directly into the distal part of the tube, and the corresponding volume of the air was evacuated proximally with the other needle.

Saline BPC 157 2 µg/rat 0 µg/kg BPC 157 2 ng/rat 10 ng/kg Saline BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg

1 month 1 month

0/0/0 4.6/7.2/13.8⁎

0.5/1.0/3.1 1.5/2.5/7.3⁎

0.5/1.0/3.5 1.1/2.1/7.5⁎

0.1/0.1/0.4 0.2/0.3/0.8⁎

89/105/140 160/230/240⁎

2.3/4.4/10.1 23.2/35.5/42.1⁎

1.2/1.6/2.5 1.8/2.7/3.4⁎

1 month

3.8/6.8/11.3⁎

1.2/2.3/8.5⁎

1.0/2.0/8.4⁎

0.2/0.3/1.1⁎

130/220/230⁎

25.3/32.5/40.6⁎

1.6/2.4/2.9⁎

% of myelinated fibers area as % of neural tissue on nerve cross section

2 months 0/0/0 2 months 17.9/26.4/38.2⁎

0.5/1.1/4.3 0.5/1.5/3.5 1.2/3.5/11.2⁎ 1.2/3.5/10.4⁎

0.1/0.2/0.6 0.2/0.5/1.3⁎

95/140/180 260/300/330⁎

4.2/6.2/11.4 46.4/54.8/59.9⁎

2.6/3.6/4.0 6.3/6.6/7.3⁎

2 months 13.3/20.4/21.3⁎

1.5/3.0/10.3⁎ 1.1/3.3/9.5⁎

0.3/0.4/0.9⁎

270/280/310⁎

40.1/51.3/53.7⁎

5.5/6.0/6.3⁎

months after injury clinically (autotomy scoring), microscopically, morphometrically and functionally (electromyoneurography, EMG), while walking analysis (sciatic functional index (SFI)) was performed at weekly intervals.

2.2. Microscopy We used part of the nerves either 5 mm distal to anastomosis, or 5 mm distal to the center of the silicon tube. Nerve samples were prepared for histological analyses by fixation in glutaraldehyde solutions, followed by post-fixation in osmium tetroxide and

dehydration in acetone. After embedding in resin, samples were cut in semithin sections (0.5–1 µm) and stained with toluidine blue. Samples were histologically assessed under light microscope for the following parameters: general histological appearance (presentation of neural fascicules, connective tissue, phagocytes, necrosis, degeneration, and edema), density and the size of regenerative fibers, existence of epineural and perineural regeneration, orientation of regenerative fibers, and proportion of connective vs. neural tissue. For morphometrical analyses light microscope camera and a special software program ISSA (VAMSTEC, Zagreb, Croatia) were used. All fascicles in each of the nerves (1–4 fascicles per nerve) were analyzed, and the diameter of myelinated fibers, thickness of myelin

Table 3 Walking patterns (sciatic functional index (SFI)) assessment throughout the sciatic nerve recovery after transection and nerve anastomosis, and therapy with stable gastric pentadecapeptide BPC 157 application. Application immediately after nerve anastomosis creation

Intra-peritoneal

Locally, at the site of anastomosis (1 ml/bath)

Intra-gastrical

Medication

Sciatic functional index (SFI) (means ± SD) weekly evaluation as analysis of the recovery of the walking patterns following sciatic nerve anastomosis Weeks following sciatic nerve anastomosis

Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg

1

2

3

− 116 ± 7

− 109 ± 7

− 107 ± 8

114 ± 6

− 115 ± 9

− 117 ± 8

4

5

6

7

8

− 95±10

− 94 ± 6

− 98 ± 10

− 86 ± 12

− 80 ± 14

− 85 ± 8⁎

− 80 ± 9⁎

− 72 ± 10⁎

− 70 ± 9⁎

− 65 ± 12⁎

− 57 ± 13⁎

− 46 ± 17⁎

− 90 ± 10⁎

− 85 ± 9⁎

− 77 ± 12⁎

− 74 ± 9⁎

− 73 ± 11⁎

− 64 ± 13⁎

− 53 ± 14⁎

− 95 ± 7

− 96 ± 8

− 92 ± 9

− 85 ± 9

− 108 ± 5

− 105 ± 6

− 100 ± 5

− 104 ± 8

− 77 ± 14⁎

− 70 ± 10⁎

− 65 ± 13⁎

− 60 ± 16⁎

− 56 ± 14⁎

− 50 ± 13⁎

− 41 ± 13⁎

− 113 ± 7

− 87 ± 8⁎

− 81 ± 6⁎

− 74 ± 12⁎

− 69 ± 9⁎

− 63 ± 12⁎

− 57 ± 11⁎

− 49 ± 17⁎

− 98 ± 9

− 97 ± 6

− 94 ± 8

− 88 ± 10

− 85 ± 7

− 120 ± 7

− 111 ± 9

− 110 ± 6

− 110 ± 10

− 80 ± 12⁎

− 76 ± 11⁎

− 70 ± 6⁎

− 68 ± 7⁎

− 63 ± 15⁎

− 54 ± 10

− 44 ± 12⁎

− 108 ± 10

− 93 ± 9⁎

− 86 ± 8⁎

− 80 ± 7⁎

− 77 ± 11⁎

− 72 ± 11⁎

− 66 ± 11⁎

− 58 ± 9⁎

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M. Gjurasin et al. / Regulatory Peptides 160 (2010) 33–41

sheet, number of myelinated fibers per area and myelinated fibers as a percentage of the nerve transected area, were measured. 2.3. EMG In separate groups of rats, with nerve anastomosis, or nerve tubing, motoneuron regeneration was assessed by the elicitation of electromyograms (EMGs) in the foot. EMGs were performed by a method modified from McDevitt et al. [41] using a TECA T 15 electromyograph apparatus. Animals were anaesthetized intraperitoneally with the administration of sodium pentobarbital (65 mg/kg). The sciatic nerve was surgically exposed and placed on stimulating hook electrodes for direct stimulation proximally or distally to the site of anastomosis or of tubing. A stimulating pulse parameter was 8 mA of threshold, 0.05 ms duration, and 1 Hz. The rat was grounded with the insertion of a fine needle percutaneously, just distal to the stimulating clips. Motor action potentials (MAP) were recorded using a pair of closely placed pins in flexor digitorum brevis in the rat foot. The MAP was amplified with filters set between 50 and 10,000 Hz using differential recording. The criteria for representative MAP were constant latency and constant amplitude upon repeated stimulation. Spinal reflexes, which were easy to distinguish due to their much longer latencies than direct recordings of EMGs and variable latencies upon repeated stimulation, were excluded. The MAP was shortly memorized in TECA T 15 electromyograph and latency and amplitude was measured. 2.4. Analysis of walking recovery

Table 4 Assessment of autotomy, a form of excessive self-care or self-grooming occurs, resulting in bite wounds and eventual self-amputation of digits. The extent of self-mutilation was assessed by counting the number of the amputated phalanges on the denervated extremity after transection and nerve anastomosis, and therapy with stable gastric pentadecapeptide BPC 157 application. Mann–Whitney U-test p b 0.05 at least vs. control. Application immediately after nerve anastomosis creation

Intra-peritoneal

Locally, at the site of anastomosis (1 ml/bath)

2.5. Autotomy

2.6. Statistical analysis Statistical analysis of the quantified data was performed by ANOVA. Post-hoc comparisons were appraised using the conservative Bonferroni/Dunn test, or non-parametric Kruskal–Wallis and subsequent Mann–Whitney U-test to compare groups. Values are presented as means ± standard deviation, or minimum/median/maximum. Statistical significant difference was considered at p b 0.05. 3. Results In general, the spontaneous course of the healing process of injured nerve was inadequate in all control rats regardless of the model used (Tables 1–4, Figs. 1A, 2A, 3A,D, 4A,B, and 5A). We demonstrated that pentadecapeptide BPC 157 improved healing of the transected sciatic nerve, either anastomozed (Figs. 1B,C,D, and 2B,C,D) or excised and nerve stumps inserted into the silicone tube (Fig. 3B,C,E,F), on postoperative weeks 4 (Figs. 1B,C,D, and 3B,C) and 8 (Figs. 2B,C,D, and

Time after nerve injury

The extent of self-mutilation as the number of the amputated phalanges on the denervated extremity Min/Med/Max

Further functional weekly evaluation includes analysis of walking patterns of the rats that expressed no or minimal autotomy as described before [27–30] using DeMedinaceli's sciatic functional index (SFI) as follows: SFI = − 38.3[(EPL-NPL)/NPL] + 109.5[(ETS − NTS)/ NTS]+13.3[(EIT − NIT)/NIT] − 8.8, (EPL and NPL-print length on both experimental and the normal side, ETS and NTS-toe spread between the first and fifth digit on both sides, EIT and NIT — the distance between the middle of the second and fourth toe on both sides).

A behavioral analysis of rats following transection of nerves to the hind leg is carried out as described previously [42,43] and self-attack of the denervated leg was observed after one or two months after injury. Autotomy, a form of excessive self-care or self-grooming occurs, resulting in bite wounds and eventual self-amputation of digits. The extent of self-mutilation was assessed by counting the number of the amputated phalanges on the denervated extremity.

Medication

Intra-gastrical

Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg Saline 1 ml/rat 5 ml/kg BPC 157 2 µg/rat 10 µg/kg BPC 157 2 ng/rat 10 ng/kg

1 month

1/5/10

1 month

0/0/0⁎

1 month

0/0/0⁎

2 months

1 /5/10

2 months

0/0/0⁎

2 months

0/0/0⁎

1 month

1/5/10

1 month

0/0/0⁎

1 month

0/0/0⁎

2 months

1/5/10

2 months

0/0/0⁎

2 months

0/0/0⁎

1 month

1/5/10

1 month

0/0/0⁎

1 month

0/0/0⁎

2 months

1/5/10

2 months

0/0/0⁎

2 months

0/0/0⁎

3E,F). While controls histologically presented with inhomogeneous pattern of regeneration, with predominantly small and middle-sized nerve fibers, necrotic detritus, and edematous connective tissue that predominated inside the fascicles, all pentadecapeptide BPC 157 rats (a single dose given locally, intragastrically, intraperitoneally or directly into the silicone tube) had more dense and homogeneous pattern of regeneration, with mainly middle sized to large nerve fibers, and marked absence tissue edema. Interestingly, unlike empty perineurium or sometimes with rare perineurial regenerates in controls (Fig. 4A,B),

M. Gjurasin et al. / Regulatory Peptides 160 (2010) 33–41

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Fig. 1. Transected sciatic nerve anastomosis. Transverse sections of distal rat sciatic nerve specimens taken 5 mm distal from the side of microanastomosis 4 weeks after operation (toluidine blue stain, 100× magnification). Saline (control) (A), stable gastric pentadecapeptide BPC 157 dissolved in saline (10 µg/kg b.w.) applied locally, at the site of anastomosis (1 ml/bath) (B), intraperitoneally (C) or intragastrically (5 ml/kg) (D), immediately after nerve anastomosis creation. Controls presented with inhomogeneous pattern of regeneration with predominantly small and middle-sized nerve fibers, inflammatory response with heavy phagocityc infiltration, necrotic detritus, and edematous connective tissue that predominates inside the fascicles (A). Pentadecapeptide BPC 157 rats (B,C,D) presented with more dense and homogeonus pattern of regeneration, with predominantly middle sized to large nerve fibers, and marked absence of inflammatory cells response or tissue edema. Histological appearance was not influenced by large vs. small dose of BPC 157.

Fig. 2. Transected sciatic nerve anastomosis. Transverse sections of distal rat sciatic nerve specimens taken 5 milimeters distal from the side of microanastomosis 8 weeks after operation (toluidine blue stain, 100× magnification). Saline (control) (A), stable gastric pentadecapeptide BPC 157 dissolved in saline (10 µg/kg b.w.) applied locally, at the site of anastomosis (1 ml/bath) (B), intraperitoneally (C) or intragastrically (5 ml/kg) (D), immediately after nerve anastomosis creation. Controls (A) still presented with inhomogeneous pattern of regeneration with predominantly small to medium size of nerve fibers, but with less pronounced inflammatory response. Pentadecapeptide BPC 157 rats (B,C,D) presented with advanced and homogeonus regeneration, extra dense intrafascicular pattern of numerous midlle size to large nerve fibers, thick myelin envelopes, and marked absence of edema and inflammatory changes. Histological appearance was not influenced by large vs. small dose of BPC 157.

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Fig. 3. Sciatic nerve recovery after transection and the 7 mm long nerve segment was dissected, the severed nerve ends were sutured to 1 mm inside a 9-mm silicone tube, and therapy with saline (A,D) or stable gastric pentadecapeptide BPC 157 (B,C,E,F) application. Representative histological sections from rats euthanized on postoperative week 4 (A,B,C) or 8 (D,E,F)(toluidine blue stain, 100× magnification). An insulin needle with a volume of 17.7 µL of the pentadecapeptide BPC 157 58 µg/ml (B,E) or 58 ng/ml (C,F) or an equivolume of the saline (A,D) was applied directly into the distal part of the tube, and the corresponding volume of the air was evacuated proximally with the other needle. Controls presented with scarce density of regenerating nerve fibers and inhomogeneous pattern of regeneration with predominantly small and middle-sized nerve fibers, connective tissue edema and inflammation, phagocytes cells and and nonhomogeonus pattern of regeneration (A,D). Pentadecapeptide BPC 157 rats (B,C,E,F) presented with markedly dense and homogeonus pattern of regeneration, with predominantly middle sized to large nerve fibers, with sporadic inflammatory cells and slight or absent tissue edema, and finally, condense pattern of axonal distribution, predominantly middle to large size haevily mielinated nerve fibers, absent inflammatory cells, and no edema.

pentadecapeptide BPC 157 rats showed an increased regeneration in perineurial fibrous tissue with large numbers of middle-sized myelinated target oriented nerve fibers, as a sign of an increased regenerative capacity (Fig. 4C,D). In all animals treated with the BPC 157 markedly faster axonal regeneration, not influenced by large vs. small dose of BPC 157 was observed. In other words, improved presentation of neural fascicles, connective tissue, density and the size of regenerative fibers, existence of epineural and perineural regeneration, orientation of regenerative fibers, and proportion of connective vs. neural tissue, all fascicles in each of the nerves showed increased diameter of myelinated fibers, thickness of myelin sheet, number of myelinated fibers per area and myelinated fibers (as a percentage of the nerve transected area) and the increased blood vessels presentation. This positive effect of the BPC 157 was recorded also as improvements in the electrophysiological (increased motor action potentials) (Tables 1 and 2) and functional

observations (markedly improved SFI index) at either 4 or 8 week period (Table 3). Accordingly, the autotomy score continually increased during the observation time in the controls with sciatic nerve anastomosis (Table 4 and Fig. 5A), while it was completely absent in the pentadecapeptide treated rats (Table 4 and Fig. 5B).

4. Discussion After traumatic injury, we presented a consistent failure of spontaneous healing of transected rat sciatic nerve exhibited in controls that was however fully counteracted by a marked improvement of healing induced by pentadecapeptide BPC 157 in anastomosed as well as non-anastomosed nerve (7 mm nerve segment resected), consistently in two different investigated models.

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Fig. 4. Transected sciatic nerve anastomosis. Transverse sections of distal rat sciatic nerve specimens taken 5 mm distal from the side of microanastomosis 8 weeks after operation (toluidine blue stain, 100× magnification). Perineural regeneration in control (A,B) and BPC 157 (C,D) rat. Empty perineurium in controls (A), some times with rare perineurial regenerates (B). Pentadecapeptide BPC 157 rats presented an increased regeneration in perineurial fibrous tissue (C) with large numbers of middle-sized heavily mielinated target oriented nerve fibers, as a sign of an increased regenerative capacity (D). Histological appearance was not influenced by large vs. small dose of BPC 157.

To evaluate the success of the applied therapy it should be noted that no carrier was used [14–19,23–25,27–30,35–40], which unmistakably permits the obtained beneficial effect to be directly attributed to the pentadecapeptide alone [14–19,23–25,27–30,35–40] (in contrast, i.e., fibrin sealant was used to deliver neurotrophic substances locally to the damaged nerve and to enhance recovery of nerve function [4]). Also, the safe application of the pentadecapeptide BPC 157 (LD1 not achieved, limit test negative, no side effects in patients [20–22]), suggests that it is different from numerous factors, like

nerve growth factor, that have been found to display severe side effects independent of the nerve injury [23]. Furthermore, it is known that the spontaneous regenerative capabilities of the nerve stumps, as well as Schwann cells abilities to provide a permissive environment for axonal elongation, are insufficient when there is a 7 mm gap between nerve ends in rat, which also presents an obstacle for the standard therapy [1]. Nevertheless, efficient healing in BPC 157 rats was repeatedly obtained (µg/ng regimens given locally, intragastrically and intraperitoneally). All these together as a summation

Fig. 5. Transected sciatic nerve anastomosis. Autotomy on postoperative week 4, a form of excessive self-care or self-grooming occurs, resulting in bite wounds and eventual selfamputation of digits (control, A). Representative presentation from rats treated with stable gastric pentadecapeptide BPC 157 (10 µg/kg b.w.) (B) applied intraperitoneally immediately after nerve anastomosis creation. Appearance was not influenced by large vs. small dose of BPC 157, or route of administration.

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of its direct and indirect effects [14–19,23–25,27–30,35–40] may indicate several possibilities for BPC 157-therapy. Generally, successful regeneration after tubulization depends on the formation of a new extracellular matrix scaffold, through which blood vessels, fibroblasts, and Schwann cells migrate to form a new nerve structure [6]. It seems that with pentadecapeptide BPC 157 therapy such permissive environment for axonal elongation is formed. Pentadecapeptide BPC 157 most probably controls the functions of collagen fragments [13,14,23–32]. Importantly, pentadecapeptide BPC 157 was more active than recombinant human platelet-derived growth factor (PDGF-BB), stimulating both expression of egr-1 and its repressor nerve growth factor 1- A binding protein-2 (nab2) [31]. Thereby, it may be important for cytokine induction, growth factor generation, early extracellular matrix (collagen) formation [31], and for the NGF signaling pathway [44]. Besides, pentadecapeptide BPC 157 has an angiogenic effect [13,14,23–32,37] and modulates NO synthesis [16,17,38,39]. These findings are very interesting, considering essential role of NO in healing [45–47], and the fact that the activation of the neuronal form of NOS has negative consequences for neuronal survival after ischemia or excitotoxicity [45], suggesting possible overriding of this negative effect by BPC 157. BPC 157's antagonization of the autotomy can also partially elucidate its beneficial effect in rats with traumatic nerve injury. It is known that autotomy reflects chronic neuropathic pain, neuroma at the proximal nerve stump, regenerative nerve sprouts growing into all directions (as seen in our control groups with severe autotomy (Fig. 5A)), with pathophysiological impulses and increased spontaneous activity in the spinal dorsal horn at the segments of projection of the injured nerve [43]. Thus, the lack of autotomy in BPC 157 treated rats with traumatic nerve injury means that it either prevented or, at least, significantly attenuated the chain of events otherwise leading to the painful sensation referred to the denervated region [48]. The noted effect of the pentadecapeptide BPC 157 application could be possibly peripheral [18] and/or central [15,19,35,36,40]. This may be particularly with respect to the evidence that dopamine diminished autotomy behavior [49] while pentadecapeptide BPC 157 can cross blood–brain barrier and counteract the central dopamine disturbances [15,19,35,36,40]. Finally, the BPC 157 protocols given shortly after injury were carefully chosen. As an illustration, the tendency is to give tacrolimus (FK506) only during a short time after injury [7,8] in small doses, 0.3 or 0.6 mg/kg, subcutaneously [9,10], which are thought to be subimmunosuppressive [11]. However, the best results are obtained at a daily, immunosuppressant, dose of 5 mg/kg in the rat [11,12]. BPC 157 on the other hand, inhibits corticosteroid induced immunosuppression [14,17,23,24,29], and given here only once and very short time after injury, had resulted in sustained and prolonged improvement. Thus, it could be concluded that pentadecapeptide BPC 157, as a stable peptide, not only positively affected wound healing [13,14,23– 32,37], but it improved the nerve healing as well. Therefore, it is likely that the waiting period before regeneration begins, the speed of the axonal regeneration, and the number of the regenerating axons are both directly (local application at the site of the anastomosis, or in tube between the nerve stumps) and/or indirectly (intra-peritoneal or intra-gastrical application) affected since the beginning. Thus, all pentadecapeptide BPC 157 regimens that were successfully given shortly after traumatic nerve injury in the present study will be suitable for further testing when given long after traumatic nerve injury.

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