alta voltagem no nervo1

7/29/2019 Alta Voltagem No Nervo1

1/7

Original a rticleISSN 1413-3555

Rev Bras Fisioter, So Carlos, v. 15, n. 4, p. 325-31, July/Aug. 2011Revista Brasileira de Fisioterapia

High-voltage electrical stimulation improvesnerve regeneration after sciatic crush injuryEstimulao eltrica de alta voltagem favorece a regenerao nervosa apscompresso do nervo isquitico

Rosana M. Teodori, Andria M. Silva, Meiricris T. Silva, Larissa S. Oliveira, Maria L. O. Polacow, Elaine C. O. Guirro

Abstract

Background: Peripheral nerve injury causes prolonged unctional limitation being a clinical challenge to identi y resources that accelerates its

recovery. Objectives: To investigate the e ect o high-voltage electrical stimulation (HVES) on the morphometric and unctional characteristicso the regenerated nerve a ter crush injury in rats. Methods: Twenty Wistar rats were randomly allocated into 4 groups: Control (CON) - without

injury and without HVES; Denervated (D) - sciatic nerve crush only; Denervated + HVES - sciatic nerve crush and HVES; SHAM - without

injury but HVES. The HVES and SHAM groups were stimulated (100 Hz; minimum voltage o 100 V, 20 s, 100 s interpulse interval) or

30 min/day, 5 days/week. The sciatic unctional index (SFI) was evaluated be ore the injury and at the 7 th, 14 th and 21 st postoperatory (PO) days.

Neural components and the area density o connective tissue, blood vessels and macrophages were analyzed. Results: Axonal diameter

was higher on the HVES than on D group, reaching almost 80% above the control values a ter 21 days (p


2/7


Introductionraumatic injury o peripheral nerves is common and

results in signi cant unctional de cits. A ter crush injuries,regeneration occurs spontaneously. In rats, no axons are oundin the muscle ten days a ter injury because the muscle rein-

nervation begins two weeks a ter the injury 1,2

.Poly-innervation is observed two days a ter the neuritis reach

the muscle, given that teen days a ter nerve crush 25% o mus-cle bers are poly-innervated. Te increase o neuritis sprouting and growth, as well as the ormation o synapses between 14 and25 days a ter injury indicate that nerve impulses may be releasedto the muscle 2. Around the 25th day, muscle membrane rest po-tential, contraction orce, sensitivity to acetylcholine, and acetyl-cholinesterase levels have returned to normal. Te process thateliminates excessive synaptic contacts starts at this time and,around the 60 th day, muscle bers become mono-innervated.Te remaining nerve terminals are increased in size and a ter 90days the postsynaptic cle t is almost completely occupied 1.

However, at the end o the regeneration morphological al-terations on regenerated axons remain. A ter section injuries,the nerve presents reduced axonal caliber and decreased my-elin sheath thickness 3. Even in crush injuries, nerve morpho-metric properties are not ully recovered4.

In humans, the most notable characteristic o denervationis muscular atrophy, which is determined by the reduction inquantity o actin and myosin myo laments, and results in a

decrease o ber diameter and loss o muscle orce5

.Lu, Huang and Carlso6 demonstrated the negative e ectscaused by an increase o intramuscular connective tissue a terdenervation, which alters muscle unction by delaying nutrientexchange between muscle bers and the capillary system, as wellas inter ering with axonal growth during reinnervation. Due toslow progress inherent to nerve recovery 7 the structural condi-tions o a denervated muscle remain widely altered during nerverecovery in humans, which compromises the return to work.Since the maturation o regenerated nerve is essential or thenerve conduction speed and unctional recovery, the main clini-

cal challenge is to identi y mechanisms to accelerate the nerveregeneration.

It is known that animal tissues are endowed with intrinsicelectricity that is involved with undamental physiological pro-cesses, such as nerve conduction and muscle contraction 8. Tecomplete motor unction recovery a ter nerve injury requiresthe occurrence o morphological and physiological processesthat determine the return o motoneurons electrical activity onthe involved muscles 9.

Te use o muscle electrical stimulation a ter peripheralnerve injury or the prevention o progressive muscle atrophy,although there is weak evidence that, can be bene cial when

applied under avorable conditions 10. Tis act rein orces the im-portance o controlled trials or the establishment o new treat-ment strategies to stimulate peripheral nerve regeneration.

High-voltage electrical stimulation (HVES) has gained ac-ceptance in both experimental and clinical research 11,12. Moststudies that use HVES investigate its circulatory 13 and regen-

erative properties on skin ulcers healing 14

.Te application o negative electric elds increases regener-

ation o the peripheral nerves in mammals 15. However, there areno studies suggesting the e ect o HVES on the peripheral nerveregeneration, which justi es the need or need or high quality studies that will permit a greater understanding o HVES andits role on this process, as well as will stimulate discussion o itsuse on the peripheral nerve injury treatment in humans. Tus,the hypothesis o the present study is that HVES can shortenthe regeneration and maturation o injured nerves.

Tere ore, the aim o this study was to investigate the in-uence o high-voltage electrical stimulation on the morpho-

metric and unctional characteristics o the regenerated nervea ter crush injury in rats.

Methodswenty male Wistar rats (210.80 10.79 g) were randomly

allocated into 4 groups (n=5 each): Control (CON) - animals without injury and without HVES; Denervated (D) - sciatic nerve

crush; Denervated + HVES (HVES) - sciatic nerve crush andHVES; SHAM - without injury, but subjected to HVES (SHAM).Tis study was approved by the Ethics Committee or Animal

Experimentation o the Universidade Federal de So Carlos (CEEA/UFSCar), So Carlos, SP, Brasil, protocol number 037/2008.

Functional gait analysis: Sciatic Functional Index (S

For the unctional gait assessment, a pathway o 8.2x42 cm was covered with white paper and the animals were set to walk on it, with their hind paws marked with ngerprinting ink. Tis

registration was per ormed prior to the crush injury, and at the7th, 14th and 21st post operatory (PO) days 16. Distances betweenthe extremity o the third nger and the calcaneous - Print Length(PL); between the rst and th toes - otal Spreading ( S); andbetween the second and ourth toes - Intermediary oes (I S) were measured in the experimental and normal paws using aMitutoyo M digital pachymeter with an accuracy o 0.01 mm,according to the manu acturers manual. Te obtained values were used in the equation proposed by the same authors 16. Teresults were expressed as unctional loss, being that the value 0(zero) representing normal unction, and the value o -100 (mi-nus one hundred) representing maximum disability.

Rev Bras Fisioter. 2011;15(4):325-31.


3/7

High-voltage electrical stimulation for nerve regeneration

Nerve injury

Te animals in D and HVES groups were submitted to acrush injury (per ormed surgically) on the le t sciatic nerve.Tey received intramuscular anesthesia with Ketamine Chlo-ride (1.16 g/10 mL) and Xylazine Chloride (2 g/100 mL) at a 3:2

ratio with doses o 0.09 mL/100 g and 0.06 mL/100 g o theirbody weight, respectively, additionally a digital trichotomy ontheir gluteal region was per ormed, bilaterally. Te le t sciaticnerve was crushed with a haemostatic orceps, 5 mm proximalto its branching point, by our 20-second clamps with one sec-ond o interval between them17. Te same researcher crushedall nerves.

Animals rom the SHAM group had the sciatic nerve ex-posed and maintained intact. All animals were kept under vivarium conditions, subjected to a 12-hour photoperiodiccycle o light/dark, controlled temperatures (23 2 C), and hadconstant access to special ood (Labina, Purina M) and water.In the rst two days a ter surgery, the animals received 4 L o Sodic Dipirone (500 mg/mL) by oral administration, or anal-gesic e ect, every 12 hours.

High Voltage Electrical Stimulation (HVES)

he Neurodyn High Volt - ANVISA 10360310008 -IBRAMEDM was used in this study. he calibration wasper ormed with an oscilloscope ektronix ( DS M) 210,

with a load o 1000 , while the timer was calibrated us-ing three stopwatches ( echnos M).Te intervention on HVES and SHAM groups was per-

ormed under anesthesia (Ketamine Chloride and XylazineChloride 0.045 mL/100 g and 0.03 mL/100 g o body weight,respectively) and was initiated 24 hours a ter the crush injury, with cathodic stimulation at the motor threshold, or 30 min-utes (100 Hz; minimum voltage o 100 V, 20 s and 100 s inter-pulse interval), 5 days a week, during 21 days. A silicon-carbonactive electrode (2.0x2.0 cm) was placed over the surgical scar, while the dispersive electrode (4.0x4.0 cm) was positioned par-

allel to the active, preserving a distance o 1 cm between them.Sterile gel was used on the skin shaved as a conductor.

Data collection

A ter 21 days, the animals were anesthetized by the sameprocedures as described or the nerve injury. Te le t sciaticnerve was xed in situ at 4 C during 10 minutes, with modi edKarnovsky xative containing 1% o para ormaldehyde and 2%o glutaraldehyde in a sodium cacodylate bu er at 0.1 M, pH 7.3.Next, the nerve segment distal to the injury was removed andthe animals were euthanized by cervical dislocation.

Te sciatic nerve ragments were maintained in the samexative solution or 24 hours and post xed in osmium tetrox-

ide at 1% in sodium cacodylate bu er at 0.1 M, pH 7.3, or twohours, immersed in 5% uranyl during 24 hours or en bloc stain-ing, dehydrated in increasing solutions (30% to 100%) o ac-etone and included in Araldite M 502 resin. ransversal section

cuts (1 m) o the distal nerve obtained 5 mm ar to the crush were stained with toluidine blue at 1% borax aqueous solution

or observation in light microscopy. In the control group, thetransversal section cuts were obtained rom the same region asthe experimental groups.

Quantitative and morphometric analysis

For these analysis the examiner was blinded. Te numberand diameter o axons and the diameter o nerve bers weredetermined 3. In this study we used an image analysis system(Image-Pro Plus 6.2 - Media Cybernetics M). From these data,the myelin sheath thickness was calculated.

For the area density analysis o connective tissue, blood ves-sels and macrophages, ve non-serial sections were selected,in which ve elds with a 1000x magni cation were randomly photographed using an In nity Lite (Lumenera Corporation M)digital camera attached to an Olympus M BX 41 light micro-scope, which was connected to an image analysis system (Im-age-Pro Plus 6.2 - Media Cybernetics M, with a 100x objectivelens). Following, a planimetry by a point-counting system18 was

used, in which a grid with 450 line intersections per eld, total-izing 56.250 intersections per experimental group, was appliedin order to obtain the percentage o area density.

Statistical analysis

Te Shapiro-Wilk normality test and a One Way ANOVA,ollowed by the post-hoc ukey test were used or the num-

ber and density o blood vessels, density o macrophages andconnective tissue, axon number, nerve morphometry and

unctional analysis. One Way ANOVA ollowed by amhanes

2 test (between-group comparisons) was used to calculateaxonal diameter. o analyze the SFI between-group di erences,

wo Way ANOVA, ollowed by post hoc LSD test were utilized.Te signi cance level o 5 % was considered or all analyses.

Results

Quantitative and morphometric analysis

No di erences on the axons number was observed in allgroups. Te axonal and ber diameter, as well as the myelin



4/7


sheath thickness, were higher on the HVES group than on theD group. Tese results are displayed in able 1.

Histological analysis

Figure 1 presents the morphological characteristics o

the normal sciatic nerve, with axons surrounded by myelinsheaths with thickness proportional to its diameter (CON). Inthe denervated group (D), the axons, as well as the myelinatednerve bers, present decreased diameters and myelin sheathsthickness, with an evident increase o neural connective tis-sue. When the injured nerve was subjected to HVES, the re-covery o their morphometric characteristics was observed. As expected, nerves that were not injured but received HVES(SHAM) presented morphological characteristics similar tonormal nerves.

Functional gait analysis

As presented in Figure 2, among the our di erent groups,the most e ective unctional recovery was observed in the ratsallocated to the HVES group at the 14 th PO day (p0.05). Macrophages and connectivetissue area densities were higher on D than on CON group

Table 1. Mean SD o quantitative and morphometric analysis on groups: Control (CON); Denervated (D); Denervated and High-voltagStimulation (HVES); Without injury but High-voltage Electrical Stimulation (SHAM).

CON D HVES SHAM

Axons Number 9161.60 1394.14 11140.60 1152.53 9780.60 2061.95 10078.00 2261.94

Axons Diameter (m) 6.73 0.38 3.53 0.43# 5.38 0.34#* 6.43 1.16 *

Fibers Diameter (m) 11.45 0.49 6.66 0.67# 11.05 0.59 * 11.48 1.21 *

Myelin Sheath Thickness (m) 1.14 0.11 0.75 0.06# 1.24 0.11 * 1.21 0.05 *

(#) Di er rom CON group; (*) Di er rom D group (p


5/7


(p


6/7


It is possible that the cathodic stimulation on motorthreshold applied at injured peripheral nerve has promoted atransient e ect on blood ow to connective tissues surround-ing the nerve, in this sense avoring the uptake o nutrients andneurotrophic actors essential to the nerves morphological andphysiological restoration, without changing the area density

and the number o blood vessels, since these morphometriccharacteristics re er to the nerve analyzed 24 hours a ter theend o stimulation.

Te results o the SHAM group demonstrated that the HVESapplication on normal nerve does not in uence its quantita-tive and morphometric characteristics, highlighting the e ecto HVES on the maturation o regenerated nerve bers.

In this study, all morphometric parameters were com-pletely recovered on the HVES group, with the exception o theaxon diameter, since it did not reach control values a ter 21days o injury. Considering that the nerve maturation is only completed when it is reconnected to the muscle and whenthe synaptic elimination occurs, which happens only aroundthe 60th day a ter crush injuries1,2 it is possible to suggest thatHVES speed axonal maturation, once that these results wereobserved only 21 days a ter injury.

Te HVES increased myelin sheath thickness in comparisonto group D, which was proportional to axon diameter and auto-matically re ected on the nerve bers diameter. Tese resultsexceeded the expectations o morphological recovery o injurednerve bers, because, according to Schrder 4, a ter crush injuries,

the axons diameter can reach control values a ter 6 months, butthe myelin sheath thickness reaches only 79% o normal valuesa ter 1 year. Mira28 identi ed the presence o regenerated nerve

bers a ter rat sciatic nerve crush between the 10 th and 15th day,however, the variation o ber diameters remained the same a tertwo years o injury. In the present study, the diameter o regener-ated axons a ter HVES treatment reached 79.9% o control values, while the nerve bers diameter reached 96.5% and the myelinsheath thickness reaches 100% o the control values. Tese resultsdemonstrate the bene ts o HVES on the maturation o regener-ated nerves a ter crush, as well as the earliness with which this oc-

curred a ter its application, emphasizing the importance o uturestudies to demonstrate its role in transection nerve injury.

Tese results are rein orced when the morphometric datao other nerve elements are examined, such as the connectivetissue. Te macrophages, responsible or cell debris phago-cytosis, presented the highest area density in D group at the21st day. In the HVES group, there was signi cant reductiono these cells, demonstrating a more advanced stage o re-generated tissue maturation. Probably, HVES stimulated themacrophages migration to the injury site because according

to Orida and Feldman 29 the macrophages tend to migratetoward the anode.

In this study, there was no in uence o HVES on normalnerve unction. On the denervated groups, the unctionalbehavior ollowed the evidence already mentioned by Carmignoto et al . 1 and Gorio et al.2, where complete unction

loss was observed between the 7th

and the 14th

day, andrecovery at the 21st PO day, when the muscle is almostcompletely re-innervated.

Te between-group analysis showed that all groups pre-sented normal unction at the preoperatory period, whereas atthe 7th PO day, denervated groups showed signi cant unctionreduction. At the 14 th PO day, these values remained close to-100 in D group, while there was signi cant unctional recovery on the HVES group at the same time period. Tis act dem-onstrates the HVES e ectiveness in accelerating recovery, notonly regarding the morphological characteristics o regeneratednerve, but also the restoration o muscles control by in eriormotoneurons. At the 21 st PO day, the two denervated groupsrecovered their unction with no di erence between HVES andD groups being observed.

Tese results suggest that the HVES use acceleratedmuscle reinnervation. Consequently, the acetylcholine re-lease on the neuromuscular junction would be occurring precociously because, according to Gorio et al. 2, a ter 15 dayso nerve crush in rats, 25% o muscle bers are reinnervated.It was observed in D group that at the 14 th PO day there was

no unctional recovery, probably due to the act that only onea ourth o muscle bers were reinnervated, which would notre ect as improved unction. However, on the HVES group,SFI values suggest that a larger number o muscle bers werereinnervated, resulting in better contraction quality and im-provement o unction.

Despite the act that the SFI results show that at the 21 st PO day all groups reached the normality values, once nerve re-generation and unctional recovery occur spontaneously a tercrush injury 7 is evident the acceleration o unctional recovery in the HVES group, rein orcing the need to investigate the

unctional parameters on nerve section injuries.It should be highlighted the importance o early unctional

recovery when considering the e ects o denervation to themuscle. A ter denervation, muscle atrophy rapidly occurs, dueto the loss o myo brillar protein components that represent60% o muscle proteins30, since contractile activity is impor-tant or the maintenance o the muscle bers cross-sectionalarea 10. Denervation reduces the muscle ber cross-sectionalarea, increases the area density o the intramuscular connec-tive tissue, undermining the muscle reinnervation, limiting the



7/7


References1. Carmignoto G, Finesso M, Siliprandi R, Gorio A. Muscle reinnervation I. Restoration o

transmitter release mechanisms. Neuroscience. 1983;8(3):392-401.

2. Gorio A, Carmignoto G, Finesso M, Polato P, Nunzi MG. Muscle reinnervation II. Sprouting,synapse ormation and repression. Neuroscience. 1983;8(3):406-16.

3. Santo Neto H, Pertille A, Teodori RM, Somazz MC, Marques MJ. Primary nerve repair by muscleautogra ts prepared with local anesthetic. Microsurgery. 2004;24(3):188-93.

4. Schrder JM. Altered ratio between axon diameter and myelin sheath thickness in regeneratednerve bers. Brain Res. 1972;45(1):49-65.

5. She fer LR, Chae J. Neuromuscular electrical stimulation in neurorehabilitation. Muscle Nerve.2007;35(5):562-90.

6. Lu DX, Huang SK, Carlson BM. Electron microscopic study o long-term denervated rat skeletalmuscle. Anat Rec. 1997;248(3):355-65.

7. Fawcett JW, Keynes RJ. Peripheral nerve regeneration. Annu Rev Neurosci. 1990;13:43-60.

8. Hess D, El Manira A. Characterization o a high-voltage-activated IA current with a role in spiketiming and locomotor pattern generation. Proc Natl Acad Sci U S A. 2001;98(9):5270-81.

9. English AW, Schwartz G, Meador W, Sabatier MJ, Mulligan A. Electrical stimulation promotesperipheral axon regeneration by enhanced neuronal neurotrophin signaling. Dev Neurobiol.2007;67(2):158-72.

10. Dow DE, Cederna PS, Hassett CA, Kostrominova TY, Faulkner JA, Dennis RG. Number o contractionsto maintain mass and orce o a denervated rat muscle. Muscle Nerve. 2004;30(1):77-86.

11. Mendel FC, Wylegala JA, Fish DR. Infuence o high voltage pulsed current on edema ormationollowing impact injury in rats. Phys Ther. 1992;72(9):668-73.

12. Garcia LB, Guirro ECO. E eitos da estimulao de alta voltage no lin edema ps-mastectomia.Rev Bras Fisioter. 2005;9(2):243-8.

13. Taylor K, Mendel FC, Fish DR, Hard R, Burton HW. E ect o high-voltage pulsed current andalternating current on macromolecular leakage in hamster cheek pouch microcirculation. PhysTher. 1997;77(12):1729-40.

14. Davini R, Nunes CV, Guirro ECO, Guirro RRJ. Estimulao eltrica de alta voltage: uma opo detratamento. Rev Bras Fisioter. 2005;9(3):249-56.

15. Romn GC, Strahlendor HK, Coates PW, Rowley BA. Stimulation o sciatic nerve regeneration inthe adult rat by low-intensity electric current. Exp Neurol. 1987;98(2):222-32.

16. Bain JR, Mackinnon SE, Hunter DA. Functional evaluation o complete sciatic peroneal, andposterior tibial nerve lesions in the rat. Plast Reconstr Surg. 1989;83(1):129-38.

17. Lima SC, Caiero QM, Peviani SM, Russo TL, Somazz MC, Salvini TF, et al. Muscle and nerve

responses a ter di erent intervals o electrical stimulation sessions on denervated rat muscleJ Phys Med Rehabil. 2009;88(2):126-35.

18. Mathieu O, Cruz-Orive LM, Hoppeler H, Weibel ER. Measuring error and sampling variatiostereology: comparison o the e ciency o various methods or planar image analysis. J Mic1981;121(Pt 1):75-88.

19. Dubov P. Schwann cells and endoneurial extracellular matrix molecules as potential cues sorting o regenerated axons: a review. Anat Sci Int. 2004;79(4):198-208.

20. Caiero QM, Betini J, Teodori RM, Minamoto VB. The e ect o time interval between elstimulation on the denervated rat muscle. Rev Bras Fisioter. 2008;12(2):143-8.

21. Oliveira LS, Sobral LL, Takeda SYM, Betini J, Guirro RRJ, Somazz MC, et al. Estimulaelctrica y natacin en la ase aguda de la axonotmesis: infuencia sobre la regeneracin nervios

y la recuperacin uncional. Rev Neurol. 2008;47(1):11-5.22. Holcomb W, Rubley MD, Girouard TJ. E ect o the simultaneous application o NMES and

on knee extension torque. J Sport Rehabil. 2007;16(4):307-18.

23. Bettany JA, Fish DR, Mendel FC. Infuence o high voltage pulsed direct current on edeormation ollowing impact injury. Phys Ther. 1990;70(4):219-24.

24. Baker LL, Chambers R, DeMuth SK, Villar F. E ects o electrical stimulation on wound heapatients with diabetic ulcers. Diabetes Care. 1997;20(3):405-12.

25. Houghton PE, Kincaid CB, Lovell M, Campbell KE, Keast DH, Woodbury MG, et al. Eelectrical stimulation on chronic leg ulcer size and appearance. Phys Ther. 2003;83(1):17-28.

26. Mohr T, Akers T, Wessman HC. E ect o high voltage stimulation on blood fow in the ratlimb. Phys Ther. 1987;67(4):526-33.

27. Gri n JW, Newsome LS, Stralka SW, Wright PE. Reduction o chronic posttraumatic edema: a comparison o high voltage pulsed current, intermittent pneumatic compression, an

placebo treatments. Phys Ther. 1990;70(5):279-86.

28. Mira JC. Quantitative studies o the regeneration o rat myelinated nerve bres: variatiin the number and size o regenerating bres a ter repeated localized reezings. J An1979;129(Pt1):77-93.

29. Orida N, Feldman JD. Directional protrusive pseudopodial activity and motility in macrophainduced by extracellular electric elds. Cell Motil. 1982;2(3):243-56.

30. Furuno K, Goodman MN, Goldberg AL. Role o di erent proteolytic systems in the degrao muscle proteins during denervation atrophy. J Biol Chem. 1990;265(15):8550-7.

31. Carter AJ, Kristmundsdottir F, Gilmour J, Glasby MA. Changes in muscle cytoarchitectua ter peripheral nerve injury and repair. A quantitative and qualitative study. J Hand Surg B1998;23(3):365-9.

interaction o nerve terminals with acetylcholine receptors atthe neuromuscular junction 31.

Te HVES application accelerated nerve regeneration, pro-pitiating to the muscle the possibility o an earlier voluntary activity recovery, thereby preventing the development o den-ervation deleterious e ects.

Concluding remarksIt is concluded that HVES accelerated the regenerated

nerve bers maturation a ter crush injury, as well as unctionalrecovery. Our results suggest that the utilization o HVES couldcontribute to a more avorable regeneration o peripheral nerve

injury by aiding in a premature unctional recovery, reducing treatment costs, and in providing a aster reintegration o thesubjects to his or her labor activity.

Additional studies may contribute to speci y the time o nerve regeneration initiation a ter HVES application, as well asto promote the use o this resource on human peripheral nerve

injury treatment.

Acknowledgmentso the Conselho Nacional de Desenvolvimento Cientfco e

Tecnolgico (CNPq), or the research productivity grant (Processn 307041/2008-5).

Rev Bras Fisioter 2011;15(4):325-31

alta voltagem no nervo1

Documents