JOURNAL OF NEUROSCIENCE

METHODS ELSEVIER Journal of Neuroscience Methods 98 (2000) 105-118 www elsevier comilocate/jneumeth

Polyimide cuff electrodes for peripheral nerve stimulation

Francisco J. Rodriguez a, Dolores Ceballos a, Martin Schuttler b, Antoni Valero a,

Elena Valderrama c, Thomas Stieglitz b, Xavier Navarro a.*

d Neuroplasticity Group, Department of Cell Biology. Physiology and Immunology. Faculty oj Medicine, Unil'er.Htat Autimoma de Barce/ona, E-08193 Bellaterra, Spain

b Department of Sensor Systems / Microsystems, Fraunhojer Instztute for Biomedical Engll1eering, St. Ingbert. Germany C Department of Computer Sciences, Universitat Autonoma de Barcelona, Bellaterra. Spalll

Received 23 August 1999; received in revised form 13 February 2000; accepted 15 February 2000

Abstract

This paper describes a new tripolar spiral cuff electrode, composed of a thin (10 11m) and flexible polyimide insulating carrier and three circumneural platinum electrodes, suitable for stimulation of peripheral nerves. The cuffs were implanted around the sciatic nerve of two groups of ten rats each, one in which the polyimide ribbon was attached to a plastic connector to characterize the in vivo stimulating properties of the electrode, and one without a connector for testing possible mechanical nerve damage by means of functional and histological methods. The polyimide cuff electrodes induced only a very mild foreign body reaction and did not change the nerve shape over a 2-6 month implantation period. There were no changes in the motor and sensory nerve conduction tests, nociceptive responses and walking track pattern over follow-up, and no morphological evidence of axonal loss or demyelination, except in one case with partial demyelination of some large fibers after 6 months. By delivering single electrical pulses through the cuff electrodes graded recruitment curves of !X-motor nerve fibers were obtained. Recruitment of all motor units was achieved with a mean charge density lower than 4 l1C/cm2 for a pulse width of 50 I1S at the time of implantation as well as 45 days thereafter. These data indicate that the polyimide cuff electrode is a stable stimulating device, with physical properties and dimensions that avoid nerve compression or activity-induced axonal damage. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Neural prosthesis; Polyimide; Spiral cuff; Functional electncal stimulation; Penpheral nerve: Passive neural damage

1. Introduction

Spinal cord injuries, due to transection or compres­sion with combined interruption of ascending and de­scending axonal pathways and local necrosis of intraspinal neurons, result in devastating functional impairment. Patients who suffer such injuries remain para- or tetraparetic for life, because of the limited capabilities for axonal regeneration and for replace­ment by new neurons within the central nervous system (Biihr and Bonhoeffer, 1994). Functional electrical neu­romuscular stimulation (FES) systems have been devel­oped in order to artificially replace the central motor control and directly stimulate the intact peripheral nerves or muscles of spinal cord injured patients, at-

* Corresponding author. Tel.: + 34-93-5811966; fax' + 34-93-5812986.

E-mail address:xavier.navarro@uab.es (X. Navarro)

tempting to generate movements or functions which mimic normal voluntary movements. There are avail­able FES prostheses for upper and lower-limb activa­tion, which allow for restoration of some basic motor functions such as hand grasp, functional stance and gait or even walking (Rushton, 1997; Heiduschka and Thanos, 1998).

Cuff electrodes may have several advantages com­pared to intramuscular, epymisial and surface elec­trodes: reduction of the stimulus intensity required for nerve activation and thus minimize hazardous electro­chemical processes secondary to charge injection and diminish the power consumption of the stimulator sys­tem, and allow for correct positioning of electrode leads to minimize mechanical distortion and the probability of lead failure. From a functional point of view cuff electrodes can be used to induce graded motor unit recruitment and muscle contraction, and make possible the selective stimulation of nerve fascicles, useful for

0165-0270/00/$ - see front matter © 2000 Elsevier Science B.V. All nghts reserved PH: SOI65-0270(00)00192-8

106 F.J. Rodri;!lIl!: e/ al. Joul'11of uJ New'o:;"('f(!IIi.'{' M('JlwJ:; 98 (2000) 105- 118

well-balanced movements or fa tigue avoidance (Fang and Mortimer, 1987; Veraart et a1.. 1993; Rushlon, 1997; Heiduschka and Thanos. 1998).

In spite of the fact that nerve cuff electrodes have been implanted in some patients for as long as 15 years (Glenn and Phelps. 1985; Waters et aI., 1985). nerves can be damaged by the presence of the cuff due to the delicacy of the nerve tissue and the physical properties of the electrodes (Nielson el aI. , 1976: Stein et aI. , 1977; Pica7-3 et aI. , 1978: Breederveld and Zilvod, 1979; Wa­ters et aI., 1985; Krarup et aI., 1989; Larsen et aI.,

a)

1998). especially in peripheral nerves subjected to a wide range of motion. CulT electrodes must be safe and stable in order to have chemical, mechanical and geo­metrical properties which do not harm the host. They have to be composed of inert materials, both passively and when subjected to electrical stimulation, since dete­rioration of the device may result in implant failure and the release of toxic products. Materials typically used are platinum, iridium and stainless steel as conductors and sil icone rubber, polytetraOuoroethylene and poly­imide as insulating carriers (Naples et aI. , 1990:

7A",

.,."'"

electrode f I II.

PI

6.75

......... Ir--

~anarcuff 5 ,

12

b)

1

'--_____ --IF :::_(PI) Spinning on polyimide (PI )

Spinning on PI ,

to be rolled

Reactive Ion Etching (RIE) of PI , removing of aluminum

6

1

1-Ribbon with interconnects

;.;iiI

= = f--

= ~

Ending enl

Pt contact pads

argement

Spinning on resist , developing, metallization, lift-off

Deposition of aluminum, wei etching

.............

Separation from wafer

Fig. I. (a) Schematic design or the polyimide cuff electrode with interconnects at a 90° angle; all dimenSIOns are In mm. (b) Schematic view of Lhe process technology for cuff electrodes wilh integrated interconnects.

F.J . Rodrigue:! et 01. I Journal of Neurosciellce Methods 98 (:!OOO) 105- 11 8 t07

Fig. 2. Micrographs of thc cuff electrode with an attached plastic connector (a}. and details of the cuff portion of the electrode (c and d). The mtcrorulcr shown in (e) is in mm. The fine line in (d) points to the overlap of the spiral cuff.

Heiduschka and Thanos. 1998). With regard to me­chanical properties, suitable cuff electrodes should be flexible and self-sizing in order to avoid stretching and compression of the nerve. the most extended designs being the helix-shaped (McCreery et aI. , 1992) and the spiral-cuIT electrodes (Rozman 1991). have a larger diameter than the nerve to be implanted (Krarup et a!.. 1989; Larsen et aI. , 1998), the shortest length and thinnest wall possible without compromising their me­chanical stability, and avoid sharp edges and blunt corners to prevent nerve damage (Naples et aI., 1990).

In this report we present the application of a new flexible. tripolar cuff electrode made of polyimide with integrated platinum contacts, and the results of its experimental in vivo implantation around the rat sciatic nerve. We evaluated the possible effects of cuIT implan­tation from 2 to 6 months on the nerve by means of histological and functional methods. and tested the cuIT electrodes for neuromuscular activation by determining the threshold and the recruitment curve of motor activity.

2. Materials and methods

2. I. Desigll 0/ rhe cliff electrode

The cuIT electrodes were made using a poly imide resin (P12611. DuPont) as substrate and insulation ma­terial (Stieglitz and Meyer. 1995; Stieglitz et aI., 1996).

Fig. 3. Micrographs of (a) one cuff electrode implanted around the rat sciatic nerve at the Ihigh. (b) a culTed nerve 2 months postimplan­lat ian, (c) the same nerve arter retrieving the cuff electrode. and (d) a sciatic nerve that was cuffed for 45 days harvested and placed in saline solution with thc cuIT elcct rode retrlcved (note the clean and vascula rized surface of the nerve I. The prox1mal di rection is at the top and the dtsta l at the bottom of the photographs. The asterisk in (al and (b) indicates the interconnect ribbon.

108 F.J. Rudrigm'; ('/ II/. )ollmal of Nmros.-icllc<' M~rlmds 98 1200fJ! 105- /18

Table I Elcclrophysiological evaluation of motor and sensory nerve fibers of the right sciall(; nerve with a polyimide cuff electrode implanted for 2 months (el; /I = 10) and for up 10 6 months (/I '" 5)"

Nerve conduction Day postlmplant

11 = 10 , . 5

0 30 60 90 120 ISO

MOlOr nen'{'

Plantar CMAP Latency 2.68 ±O.09 2.65 ±O.O7 2.76 ±O.O7 2.59 ± 0.05 256 ± 0.05 2.63 ± 0.05 Amplitude 8.l8±O.16 7.79 ± 0.21 7.79 ± 0.73 8.17±O.72 7.66 ± 0.62 7.98 ± OJ)

Plantar H-wavc Latency 7.19±O.\3 7.34 ±O.2J 7.36±O.16 6.74 ±O. 11 6.56 ± 0.14 6.67 ± 0.23 Amplitude O.87±O.14 O.68±O.19 I. IO±O.23 O.7S±O.17 0.87 ± 0.2 1 1.37 ±O.40

Gast rocnemius CMAP Latency 1.43 ±O.OS 1.47 ± O.04 1.46 ±O.04 1.38 ±O.O7 1.38 ± 0.06 1.43 ± 0.05 Amplitude 43.7 ± 1.00 45.3 ± 1.53 45.8 ± 1.20 45.8± 1.5 46.3 ± 1.2 44.0 ± 0.9

Sensor), nerve Digital CNAP Latency 1.88 ± 0.05 1.92 ± 005 1.93 ± 0.06 1.90 ± 0.06 1.90 ± 0.01 1.92± 0 08

Amplitude 19.2 ± 0.10 19.2 ± 0.60 18.2 ±0.1I0 17.6±0.88 I1.S ± 1.10 11.2 ± 1.70 Tibial CNA P Latency 1.02 ± 0.04 1.03 ± 0.04 1.08 ± O.04 1.00 ± 0.04 1.03 ± 0.03 1.06 ± 0.04

Amplitude 196 ± 5.8 119 ± 6.1 175 ± 5.2 118 ± 7.3 180 ± 10.9 176 ± 99

~ Latency in ms; amplitude in mV for CMAPs and in IlV for CNAPs. No significant dltTcrenccs between any mtervals. paired I-test.

;;-!" • • ~ Q. S5 • • • • • 4S u • • .~

_ L _-~-

i • " • • • • • C

" 1O+-~--.----_, 0 r ____________ ~ ___________ ro ______ o ________ _C~C_ ______ _,w

I ~ a li.I --+-- an --+-- .u ~ .ql -.........- avl

! --0--- .... 1 ---<>--- azl ---0--- .d --6-- azl ----9-- az4 , 4 2.5

1.5 '" ! • ~ • 2 •

3 :l • • ~ "'--:::x • i:::' • 2.' z u

• a • _1 .5

2 ~ Q

• • r. 1.5 1

o 30 ro o DAY POST- IMPLANTATION DAY POST-IMPLANTATION

Fig. 4. Individual I;hanges over a 2 month follow-up in motor and sensory nerve rondul;l1on parameters of the I;uffed sciatic nervc.

FJ. Rodrigu/'; ('/ al. Jourl/al oj N('uru.~cie/l("(> Mc{hods 98/200u) /U5 - /18 109

It consists of a Hal rectangu lar piece (1 2 x 6.75 mm). an intercon nect ribbon al a 90° angle (2 mm wide. 26 mm long) and an ending enlargemen t (2 x 10 mm) with contact pads (Fig. Ja). The whole structure has a thick ness of 10 J1ffi and a weight of 5 mg. and is highly

fl exible. The cufT part is ro lled to become a cyl inder with an inner diameter of 1.7 mm and a length of 1.2 mm (Fig. 2). Three recording-stimu lat ing plat inum elec­trodes are arranged with a 5 mm interelectrode distance.

Table 2 Functional evaluation of walking pancrn and pam ~n.ibihty in ratS with a polYlmide cuff electrode implanted around the right sclatl\: nerve for 2 months (el: 11 = 10) and for up to 6 months (11 = 5)'

Days posllmplanl

n = 10 11 = 5

30 60 90 120 ISO

W"lkilig {ruck I'rint length ( 'In R/L) 101.8 ± 3.01 101.1 ± 2.22 10U ± 10 96.1 ± 2.-I 96.b ±4.2 Toe spreading e% R/L) 98.4 ± 2.42 IOO.9 ± 1.39 102.3±2.7 99.5 ± 5A 99.~ ± 21

Scintic functional indcx - 2.12±2.-I2 4.1 ± 7.05 6.0 ± 6.3 1.6±8. 1 -I2±H

Plal!lar a/gcsilll/'Iry Threshold ("C). R 40.1 ± 0.3 39.8 ± 0.2 40.5 ± 0.9 -I1.6 ± 04 40.5 ± 0.3 Threshold (0C). L 40.7 ± 0.7 -11.3 ± 0.5 40.4 ± 0.7 -I1.8 ± 0.; 41.0 ± 0.3

.• No Significant differences between any Intervals. p:ured Hest

Table 3 Morphological f"valuation of transverse seclions laken al the muJpoint and distally to a polyimidc culT electrode implanted around the TIght SClUtic nerVf"S for 2 months (/1 = 5) and for 6 months (n = 5) in comparison" Ilh correspondmg levels of the Intact contralateral sciallc nerves (11 = 5)"

Ncrvc cross·sectlon area (mm~) DenSity of myelinated fibers No. of myclinated fibers

Right (cuff) Left (control) Right Icum Lct"t (comrol) Right (cuff) Left (cOlltrol)

Mid Distal Mid Distal Mid Distal Mid Distal Mid Dlslal Mid Distal

lmfll Ral akl 0.576 0.646 0.828 0665 13771 13180 9S54 12436 7925 8512 7912 8270 Rat aU 0.987 0.572 0.646 0.89 8833 14617 13384 10022 8716 8362 8649 8921 Rat all 0.643 0.676 0.861 0.464 11599 12000 10333 17801 8106 8106 8895 8263 Ral al2 0.734 0.701 0.618 0.441 10833 10561 13450 13535 7947 7398 8305 64]3 Ral al3 0.631 0.498 0.586 0.539 14329 12861 14493 16456 9f).i' 6804 8487 8873

Mean 0.714 0.618 0.708 0.600 11073 12644 I 22·B 1-W50 8347 7836 8450 8152 S.E.M. 0.073 0.037 0.057 0082 1007 670 %7 1395 226 321 166 452

6 mpi Ral3vl 0.634 0.5JO 0637 0.526 13946 15406 13150 16775 8838 7863 844-1 8827 Rat awl 0.695 0.898 0.645 0.888 11831 10085 13970 10295 8219 9051 9016 9146 Ral azl 0.629 0.662 0549 0.789 I,,"' 13545 15176 9649 9062 8963 8332 7613 Rat az~ 0.625 0.580 0.491 0597 14353 15746 17077 13011 8968 9134 8387 7769 Rat a7A 0.712 0.758 0.602 0.710 123J 1 10027 14050 12030 8767 7603 8458 8541

Mean 0.659 0.682 0.585 0.702 I.U69 12962 14705 12352 8771 8523 8527 8379 S.E.M. 0018 0.068 0.029 0.065 541 1244 668 J257 147 326 114 298

Morphometry parametcr Level 2 Monlhs poslimplant 6 Months postunplant

Right Icum Left (comrol) Right (cuff) Left (control)

No. degcneratlng fibers Mid 39 ± 39 0 OS ± 0.8 0 No. demyelinated fitx-rs Mid 0 0 24.2 ± 2J.S 0 Axonal diameter \j.lm) Mid 4.67 ± 0.04 4.58 ± 004 4.41 ± 0.03 4.52 ± 0 03 Myehn thickness Ij.lm) fo,1Id 1.64 ± 001 1.68 ± 0.01 1.61 ± 0.01 J.61± 0.01 g RatiO Mid 0662 ± 0.001 0.648 ± 0.001 o (,..l7 ± 0.001 0.650 ± 0.00 I

" No Significant changes between palrell nerves: Wilcoxon signed rank lesl.

11 0 F.) , Rodriglfe; e/ ,,/ JUI/mal uJ Nl!llro.~( ·I(,II(,(· ;\Ier/lOtis 9R (200m I05- JJS

Fig. 5. Transverse sections of the whole sciatic nerve (a) and details of the morphology of the myelinated fibers at the level of the cuff electrode (c) and distally (e) after 2 month implantat ion. Control samples sectioned at the same levels are shown in the right column (b. d, I), Bar = 250 J.lm in a, b; bar = 10 J.lrn in c-f.

The fabrication of the polyimide-based microstructure with a single metallization layer has been described in detail elsewhere (Stieglitz et aI., 1997). First a layer of polyimide resin (5 ~m) is spun onto a silicon wafer used as a support structure (Fig. I b (I ». The bottom layer can vary between 2 and 7 ~m, with a usual thickness of 5 ~m. The polyimide is dried and imidized in a curing process at 350°C under nitrogen atmosphere. In a second step thin film technology was used to creatc the platinum electrodes, multi-strand interconnects and contact pads in a single process step. Arter curing the polyimide, the metall ization (30 nm titanium, 300 nm platinum) for the contact pads and the interconnect lines in the ribbon and electrode sites was deposited by sputtering and structured in a lirt-offtechnique (Fig. I b (2». A polyimide top layer of 5 ~m thickness was spun on and cured to provide additional mechanical support and for insulation of the metallization (Fig. I b (3». and an aluminum layer was deposited and structured as an etching mask for the following process step (Fig. I b (4». Reactive ion etching (RIE) was used to open the sites where the platinum must remain exposed, i.e. the electrode sites and the contact pads. and to separate all devices by etching the outer

shapes down to the support wafer in one etching step (Fig. I b (5». After removing the a luminum, the single devices are then separated from the support wafer (Fig. I b (6)). The planar devices were inserted , rolled and fixed in the final culT-shape in a temper step at 340°C for 2 h, by using a specially designed tool. After the temper step, the culTs were released from the tool and remained stable in a cylinder spiral shape, with an overlap of about 1.4 mm (Fig. 2). Some culT electrodes were connected to standard plastic connectors (4 x 10 mm) with a pitch of 1.27 mm with conductive glue (Epo-Tek H20E-PFC, Polytec. Waldbronn, Germany) (Fig. 2a). The connec­tion site was stabilized with small pieces of ceramics and encapsulated with silicone (Q 1-9239. Dow Corning, Wiesbaden. Germany) . The plastic connector was plugged into a male connector (8 pins) and a nat ribbon cable connected to an external electrical stimulator for delivering stimulation through the culT electrode.

2.2. ExperimellIal design

Two batches of culT electrodes made orpolyimide have been used: culTs without and with an attached plastic connector. All the culTs had the same physical

F.J. Rodrigue: 1'1 al . .. Joumol oj Nt'urosrienIT Mt'thods 98 (2fXXJ) /05- //8 '" dimensions (1.7 mm i.d. ; 12 mm length) and a 26 mm long ribbon with integrated interconnections. Surgical implantation was perfo rmed in female Sprague- Daw­ley rats under pentobarbi tal anesthesia (40 mg/kg i.p.) with the aid of a dissecti ng microscope. The right sciatic nerve (with an average diameter of abo ut 1.2 mm) was exposed at the midthigh and carefully freed from surrounding tissues from the sciatic notch to the knee. The cufT was opened and placed a round the sciatic nerve avoiding compression and st retch (Fig. 3). Arter release the spiral cuff closed covering the whole nerve perimeter, to which it adhered by surface tension. The electrode interconnect ribbon was routed through the muscle plane excision, avoidi ng tension. and the ending enlargement without or with an attached plastic connector placed subcutaneously on the upper lateral side of the hindlimb. A loose suture stitch was placed

Control neN.,

25

20

~ 15 , : 10

5

0 "1-0 2 • • • 10 12 " Axon diamel ... ijwn)

•

around the distal part of the ribbon to avoid displace­ment. The wound was closed by planes with 2-0 sutu res and d isinfected.

In one group of rats (C l , 1/ = 10) cufT electrodes were implanted around the sciatic nerve. These rats were evaluated month ly by non-invasive neurophysiological tests to obtain evidence of possible damage by the implanted cuff electrode on the nerve . By 2 months postimplan tation in five rats and by 6 months in the remaini ng fi ve rats, the nerve- cufT ensemble was re­trieved and processed by histological methods to quan­ti tatively evaluate the myelinated fibers. In a second group of rats (e2, II = 10) cufT electrodes attached to a plastic connector were similarly implanted. These ani­mals were subjected to functional tests, and also mea­surements of motor threshold and recruitmcnt curvcs were made immediately after implantat ion and 35- 45

•• •• .7

• •• - ... . . . . • . ...... : ow ...... __ ....

t" ~ .. - ..... ~ •

~ .• ..~ .; ~~ •.. .;.. .

~ .5 .\ ... -.. ~' •• • .3 .2

0 10 20 30 50 Axon pefime1er (JJm)

Cuffed nerv.s 2 month.

25 ,---------, 20

~ 15

: 10

o 2 4 6 8 10 12 14 Axon clamet ... ijlm)

•

1

•• •• .7

g .6 ~ .5

•• . 3

. ". . ~. ~i1ii· ytf,. :.:-. . . •

.2 +-~~~-~~~~,.....I o 10 20 30 40

Axon perimelflr (JJm) 50

Cuffed neNes 6 month.

25 ,------------------,

20

~ 15

: 10

o 2 4 6 8 10 Axon diameter (vm)

12

•• •• • . 7 .g .6

£ .5

•• . 3 • •

.. • • •

.2 -j-.-~r_.....,~~~~~~.-l o 10 20 30 50

Axon perimetet ijlm)

Fig. 6. Histogram distribution of myelinated axon diameters and plots of the g ratio/fiber perimeter relationship for the lcft intact sciatic nerves and for the right SCiatic nerves of ratS w1\h a polylmide euff elcct rodc Implanll-d for 2 and for 6 months

112 F.J. Rodriglle: e( (lJ. :' Jol/rl/aJ 0/ Nellrosciellce Methods 98 (2000) 105- JlS

* *

Fig. 7. Micrographs of the inner surface of the polyimidc cuff electrode after explantation of cuffs Implanted around the sciatic nerve for 2 months. (a) Under direct microscopy note the smooth and translucent surface of the polYlmidc. (b, c) Under scannmg electron microscopy no stnlctural damage was found. Asterisks indicate the platmum electrode.

Table 4

days thereafter. by stimulaling through the cuff elec­trode and recording electromyographic signals from the plantar muscles.

2.3. Functional methods

The rats were repeatedly evaluated before and up to 6 months postimplantation by non-invasive tests in order to assess possible changes in distal target innerva­tion by the sciatic nerve . For nerve conduction studies, the scia tic nerve was stimula ted percutaneously thro ugh a pair of needle electrodes placed at the sciatic notch. Rectangular elewical pulses (Grass S88) of 0.05 ms duration were applied up to a voltage that gave a compound action potential of maximal amplitude. The compound muscle action potentials (CMAPs). elici ted by orthodromic conduction of stimulated motor nerve fibers (M wave) and by the monosynaptic refiex arch (H wave) el icited by stimulation of large la sensory fibers, were recorded from the third interosseus muscle and from the medial gastrocnemius muscle with small needle electrodes inserted in each muscle (Navarro et aI. , 1994, 1996). Similarly, the sensory compound nerve action potential (CNA P) was recordcd by electrodes placed percutaneously near the digital nerves of the fourth toe and the tibial nerve at the ankle (Navarro et aJ.. 1994). The evoked act ion potentials were displayed on a storage oscilloscope (Tektronix 222 1) at settings appropriate to measure the amplitude from baseline to peak and the la lency to the onsct. During electrophysi-

Values of stimulus intensity and charge injcction for threshold and maximal ampli tude of the plantar CMAP for the four pulse duration tested (I. 0.5. 0.1 and 0.05 ms) at the day and after 35-45 days of cu ff Implanlalion4

Duration (ms) Threshold

Day 0 I

0.5

0.1

0.05

45 Days I

0.5

0.1

Stimu lus current (~A)

109 t 27 (45-340)

119 ± 26 144-336)

255 ± 31 (122--448)

410 ± 46 (240-672)

127 ± 23 (44-232)

lJ3 ± 28 140-304)

296 ± 72 (48- 840)

Charge/pha~ Charge density I~C) I~C/'m')

0. 11 ±0.03 5.77 ± 1.41 t2.4-t8) 10.0S-<l.34) O.06tOOt 3. 17 ± 0.68 (1.2- 8.91 (0.02- 0.17) O.oJ ±0.01 I 35± 0.16 (0.6-2.4) (O.Ot-O.04) O.OHO.OI 1.09 ± 0.12tO.6-1.8) (0.01-0.03)

0.13±0.02 6.76± 1.22 12.3- 12) (0.04-0.23) 0.07 ± 0.01 3.53 t o.75 (I 1- 8. t ) (0 .02- 0.15) 0.03 ± 0.01 1.58 ± 0.38 (0.3-4.5) (0.01-0.08)

0.05 486 ± 116 (66-1400) 0.02 ± 0.01 1.29 ± 0.31 to.2- 3.7) (0.01-0.07)

a Minimum and maximum values betwcen parenthesis (C2: ,,= 10).

Maximal recrullment

Stimulus current (IlA) Charge/phase (Ile) Charge densi ty I~Cicm')

322 ± 50 ( 110-624) 0.32 ± 0.05 (0. 11- 0.62) 17. 15 ± 2.64 t5 .8- 33)

310 ± 43 (92- 536) 0.15 ± 0.02 (0.05- 0.27) 8.24 ± 1.15 (2.4-141

483±70 (170-8 16) O.OS! 0.01 (0.02-0.08) 2.57 ± 0.37 ( 0.9-4.3)

788 ± I2J t308-(560) 004 ± 0.01 (0.02-0.08) 2.10 ± 0.33 (0.8-4.1)

306 ± 55 196- 564) 0.3 1 ±0.05 (0.I()-o.56) 16.27±292t5.1-30)

399 ± 130 (68- )480) 0.20 ± 0.07 (0.03-0.74) 10.62 ± 3.46 (1.8- 39)

909 ±474 (70-5120) 0.09 ± 0.05 (0.01-0.51) 4.84 ± 2.5210.4-271

1227 ± 568 196-6240) 0.06 ± 0.03 (0.01-0.31) 3.26± 1 51 10.3- 17I

f:J. Rudrigue: e/ (II. ; Journal oj Neurosf/Cllce Me/hods 98 (1OQO) 105- 118 113

1600

14" a

~ '20' '00'

~ ~

.00

• e 60. ~ 40.

200

• • '.2 0.4 0.6 ••• Pulse width (msec)

b

• Pulse width (msec)

Fig. 8. Mean thresholds for a pulse wIdth of I. 0.5. 0.1 ~nd 0.05 ms at (a) the day of implantation and (bl 35- 45 days after (group C1. 11 = 10).

ological tests the animals were placed over a wa rm flat streamer controlled by a hot water circulating pump. and the hindpaw skin temperature was maintained above 32°C.

Nociceptive responses were eval uated by a heat-radi­ation method using a modified plantar algesimeter (Hargreaves ct al., 1988). The rats were placed into a plastic box with an elevated glass noor. From the boltom of the box the light of a projection lamp was focused directly onto the plantar su rface of one hind­paw. The time spent unt il raising the heated hind paw was measured through a time-meter coupled wi th IR detectors d irected to the plantar surface. The threshold temperature for withdrawal was calculated from cali­bration curves made each testing day. The value for a test was the mean of three trials separated by 30 min resting periods.

For the wa lking track test the plantar surface of the hindpaws was impregna ted with acrylic paint and the rat placed in a narrow corridor with white paper al the base. The rat walked through the corridor to a dark compart ment at the end, leaving the footprints on the paper. The plantar length. the distance between the first

and fifth toes and between the second and four th toes were measurcd on the footp rints, to construct an index of the walking pattern of the implantcd hind paw with respect to the contralatera l intact hind paw (De Mcdi­naccli el al.. 1982).

2.4. Eleclrophysiological testillg oj the cuff electrodes

In rats with cuff electrodes attached 1O a plastic connector a protocol for characteriza tion of the elec­trode performance was applied immediately after im­plantation and again after 35- 45 days. The cufTed nerve was stimulated wi th the centra l ring electrode as cathode and the two outer ring electrodes as anodes. Current-controlled single rectangula r pulses of increas­ing intensity were deli vered from a Grass S88 stimula­tor with a PSI U6 unit to determine the threshold and the rccruitment cu rves of ex-motor fibers for pulses of 0.05, 0.1. 0.5 and I ms duration. The response was recorded electromyographically from the plantar mus­cles while the stimulus intensity was measured simulta­neously in the ot her channel of the oscilloscope.

2.5. Histological methods

Ten days after the final fu nctional studies the rats werc anesthetized and the operated area dissected. The cuff was carefully freed from the surrounding fibrous tissue and opened. The sciatic nerve was fi xed by immerSIon in glu taraldehyde- paraformaldehyde (3%:3%) in cacodylate buffcr (0.1 M, pH 7.4) for 4 h al 4°C, then. post-fixed with 2°;', osmi um tetroxide. washed in distilled water. dehydrated in graded concen­trations of ethanol and embedded in Agar 100 resin. Light microscopy observations were performed on 0.5 J1m scmirhin sections stained with toluidine blue, under an Olympus BXAO microscope. Morphomet rical eva lu­ation. including nerve cross-sect ional area, axonal counts. myelinated axon and fiber perimeters and di­ameters, was made from systemat ically selected fields at 2000 x magnification covering at least 1000 myelinated fibers (15- 20'% of Ihe nerve transverse area). with the help of a computer linked digitizing table and soft ware designed for a Macintosh (Gomez el al.. 1996). Thc densi ty and total number of myelinated nerve fibers and the g ra tio were then derivcd . Ultrastructura l observa­tions were performed on ultrat hin sections (90 nm) collected on Form var film coated hole grids and stained with lead citrate. viewed under transm ission electron microscopy.

All data are expressed as the mean and S.E. Com­parisons between runctional results obta ined at differ­en t fo llow-up times for the same animals were made by the paired I-test, while comparisons of morphological parameters were made by Wilcoxon signed rank and Kolmogorov- Smirnov tests.

114 F.J. Rodriglle; <'I ilf JVllrlwloJj NrufQS<'I<" w/' ,\/,'llw'/',' 98 (201J0) /OS- 118

3. Results

3.1. Effects of po/yill/ide cliff electrodes imp/allied around the scia/ic nerve

Over the 2 month implantation lime, there were no significant changes in any of the parameters of motor and sensory nerve conduction lests performed in groups Cl and C2. The five rats of group CI which were evaluated over a 6 month follow- up did not show any significant change either. The latency and the amplitude of the CMAPs and the CNA Ps obtained by stim ulation of the right scia tic nerve before cufT electrode implan ta­tion did not change notably at any interval postimplan­tatian (Table 1, Fig. 4). The eleclrophysioiogicai va lues were similar to those obtained in unoperated control rats tested monthly in our laboratory. The walking track measurements did not show variations between the right and left hind paws over fo llow-up (Table 2). The plantar algesimetry tests yielded simi lar values of the hot pain threshold for withdrawal. without evidence of hyperalgesia that might be induced by the loosely fitting culT (Mackinnon et aI., 1984).

At the final dissection the polyimide euIT and ribbon appeared covered by thin fibrous tissue, well vascular­ized and in con tinuity with the epineu rium. This cap­sule was constituted of connective tissue and fibroblasts. In a ll the animals the culT remained in site com pletely surrounding the sciatic nerve perimeter (Fig.

day of implantation

~ 8000 ~ - 6000 ..

0(

" '000 0

!! 2000 c • iL l~ 0

a

0 100 200 300 <00 500

1-- 1m. ---<r- 0.5 ms

;; 8000

.2! 6000 .. 0(

" .. 000 0 " • - 2000 c

~ c 0

0 100 200 300 <00 soo

Stimulus ({.lA)

3). After carefu lly cutti ng the fibrous capsule, the cuff was easily opened to free the nerve: the cuff- nerve interface was filled with extracellular fluid with no adherences or fibrosis. Transverse semi thin sections of the cuITed nerves, studied under light microscopy, showed that the general morphology, the cross-sec­tional area of the cuffed nerve, the density and the estimated total number of myelinated fibers were simi­lar to those of the intact contralateral nerves (Table 3, Fig. 5). There was a low number (2.5%) of myelinated fi bers with signs of degeneration in only one of the nerves (rat ak3) examined at 2 months, and pa rtial demyelination of some large fibe rs (1.1 %) in another nerve (rat az4) examined at 6 months postimplantation . The morphometrical measurements of axonal diame­ters, myelin thickness and g ratio were also equivalent between right and left sciatic nerves. The histogram distribution of axonal caliber and the plots of g ratio versus axonal perimeter did not show any quantitative change (Fig. 6). Under elcctron microscopy there were no abnormalities observed in the myelin sheaths, or­ganelle structure in the axoplasm and unmyeli nated fibe rs in cuffed and control nerves.

The ex plan ted polyimide culT electrodes were thor­ough ly viewed under the d issection microscope and images of the inner cufT surface laken under scanning electron microscopy. No evidences of cracking or de­lam ination of the structure were obscrved (Fig. 7).

37 days post-impJantation

8000

o 200 400 600 800 1000 1200

____ 0.1 ms --0-- 0.05 ms I 8000

o 100 200 300 400 SOO

Stimulus ({.lA)

Fig. 9. Motor recruitment curves for all stimulus duration tested at the day of implantation (a. c) and 36- 37 days after implantation lb, d) for two representative animals, one with increasing and one with decreasing thresholds with respect !O the day of Implantation .

FJ. RodrigUt>= ,·1 (/1 . . ' Journal of NeurosCIence Mt>lllOds 98 (2000) 105- 118 lIS

3.2. Properlies of Ihe poiyimide cuff elecrrodes for nerve Slimll/aliOfl

St imulation of the sciatic nerve through the tripolar cuff electrode gave similar results to stimu la tion with monopolar percutaneous needles for the gastrocnem ius and plantar CMAPs ampl itude obtained immediately and 45 days after implantation, thus indicating full activation of the a-motor nerve fibers run ning in the sciatic nerve branches to these muscles. The H wave reflex was also elici ted to a similar amplitude by either cufT or needle electrodes, indicating adequate excitat ion of propioceptive sensory fibers.

The stimulus current necessary to induce a motor response va ries wi th the different stimulus durat ion tested, being similar for I and 0.5 ms and progressively higher for 0.1 and 0.05 rus (Table 4, Figs. 8 and 9). On the day of implantation, the mean threshold for I rus stimulus duration was 109 ± 27 I-IA, and the mean stimulus intensi ty to evoke the maximal motor response was 322 ± 50 J.lA. The former va lue yields a mean charge injection per phase of 0.32 ± 0.05 IlC and a mean charge density injection of 17 ± 2.6 IlA/em", be­ing the maximal stimulus charge injected 0.62 I-IC/phase and 33 IlCfem2. For the shortest stimulus duration, 0.05 ms, the mean threshold was 410 ± 46 IlA and a maxi­mal motor response was achieved at 788 ± 123 IlA, tha t represents 0.04 ± 0.01 IlCfphase and 2.1 ± 0.33 pC/em; .

After 35- 45 days of the cuff electrode implanta tion. the threshold increased and the recru itment curves shifted to the righ t in four anima ls, decreased in five and remai ned unchanged in one anima l in comparison with the results obtained on the day of implantation. The mean thresholds for the diffcrent durations in ­creased by 11- 18% with respect to the day of implanta­tion, while the stimulus intensity fo r maximal response increased notably for the short duration stimuli of 0. 1 and 0.05 ms (Table 4). In tenus of charge density, the increase of the mean thresholds is mostly auri buted to one animal which needed 39 ~lC/em1 with a stimulus duration of 0.5 ms, 27 IlCfcm2 wi th a stimulus dura tion of 0. 1 ms and 16 IlC/cm2 with a st imu lus duration of 0.05 ms, whi le for the other nine rats the stimulus charge did not exceed va lues of 14. 4 and 3.3 ~C/cm2, respectively.

4. Discussion

For optimization of FES biomedical appl icat ions it is mandatory to know the immediate and long-term struc­tural and functional responses of the nerve to the implanted dcvice, and in part icular to know how these changes affect the electrode/neural interface. In the present study we show that the implantation of a spiral-cu ff electrode of highly flexible polyimide con-

taining three integrated circumneural platinum elec­trodcs, attached by a thin polyimide ribbon to an external plastic connector is functiona lly and histologi­ca lly harmless for the nerve after bei ng implanted for 2 months and allows for efficien t stim ulation of all motor un its wi th a mean charge density of less than 4 llC/cm1

at the time of implantation and after 45 days of implan­tation for a pulse width of 50 J1s.

The stability of the materials in the cuff electrode is crucial since, once implanted , it should remain within the body of the patient fo r many yea rs. Thus, it has to be resistant to corrosion during stimulation and to the attack of biological fluids , enzymes and macrophages, produced duri ng the ini tial foreign body reaction. Oth­erwisc, degradation of the materials and subsequent fragmentation of the device will lead to implant failure. Polyimide has deserved attention during the last few yea rs for encapsu lation or as insu lating carrier material because of its biocompatibility and nexibi lity (Richard­son ct ai. , 1993; Stieglitz and Meyer. 1995: Rihova. 1996). The polyimide PI2611 used in our device has been shown by ou r prescnt and previous results (Navarro et aI. , 1998) to be Hexible but not fragi le, as it was intact up to 6 months after implantation, and on ly generated mild fibrous reaclion aftcr in vivo implanta­tion. The new generation of polyimides, with low water absorption. has overcome some problems previously reported from the biological use of polyimide elec­trodes, such as the appearancc of stress crack ing and the tendency to collect water that results in an increase in the volume of the substratc (Locb and Pcck , 1996: Gonza lez and Rodriguez. 1997). On the other hand. platinum is the most commonly used electrode material , and although under certain conditions of stimulation it may sufTcr a small corrosion rate with potential neuro­toxic effects (MeHardy et al.. 1980: Roblee et al.. 1983), it has been extensively proved to be stable and inert and to release a negligible amou nt of platinum ions into the surrounding tissue even after long periods of stimula­tion (Heid uschka and Thanos, 1998).

One of thc main problems in the design of cuff electrodes is determination of their optimal physical properties and dimensions in order to avoid passive mechanical damage to the nerve. It is important to provide cuff flexibility to minimize secondary lesions caused by repeated stretching and rubbing of the nervc over the cuff duri ng locomotion. specially ncar the joints. Implan tation of the polyimide cufTs around the rat sciatic nerve for up to 6 months did not cause signs ofaxonopathy. axonal loss or demyelination, as evi­denced by our funct ional and histological resu lts. This is attributable to the thin wall of the polyimide cuff electrode. that reduccs the volume of the device, avoids tension by the leads and maintains a stable inner cufT d iameter. Second ly, wc used a spiral cuff with an internal area about two times larger than the cross-sec-

116 F.J Roc/rigll('; ('I (// , JOllrnol u) ,V"llrl)J'l"It",{(, .Ilelhods 98 C!(}()()) /U5 - 118

tional area of the nerve. as recommcnded from experi­mental studies on tube repair for ne rve regeneration (Ducker and Hayes, 1968; Buti et al.. 1996). With this relationshi p. the cuff initia lly adheres to the epineu rial cover by surface tension but later compression is avoided. Although snug cuff electrodes have been advo­cated to reduce the stimulus charge injection o r to obtain a high signal-to-noise ratio for ne ural recordings (Naples et al.. 1990; Larsen et al., 1998). different studies have shown that chronic implan tation of snug cuffs modifies the nerve shape lGrill and Mortimer, 1998; Larsen et al.. 1998) and produces a loss of large ncrve fi bcrs, which are the most sensiti ve to compres­sion (Mackinnon et al.. 1984; Krarup et al.. 1989; L,:lfsen et al.. 1998). Self-sizing silicone culT electrodes with a helicoidal (McCreery et al. . 1992) or a spi ral shape (Na ples et al.. 1988). which a re relatively adapt­able to possible nerve trunk volume changes due to postoperative edema. did not produce significant me­chanical injury when im pl anted without elcctrode \cads up to 4 (Agnew et al.. 1980) and 14 months (Naples et al., 1988) in cats, but when implantcd with elect rode leads, moderate nerve damage was found (Naples et al.. 1990), likely due to the additional tension applied by the leads to the cuff.

Any potential FES device should gua rantee a stan­dard functional ity and be manufactured using repeat­able processes. This results in homogeneous electrodes with a known surface. able to provide nerve stimu lation below the charge-ca rrying capacity and density that induce reversible electrochemica l processes and axonal damage (Brummer et aI., 1983; Naples et al .. 1990; Loeb and Peck. 1996). The polyimide cuff electrode tested gave a relat ively homogeneous stimulus intensity fo r threshold and maximal activation of motor nerve fibe rs belween animals (Table 4). With a stimulus dura­tion of 50 I1S we wcre able to stimulate the fi rst motor axons with a mean of 20 nCfphase and all motor nerve fi bers with a mean of 40 nC/phase on the day of implan tation and of 60 nCfphase after 45 days, with the maximum stimulus needed of 80 nCfphase except fo r one animal with 312 nCjphase. These values are well below the margins for safe repet itive st imulation deter­mined by McCreery et a l. (1 992) at 150 nC/phase . On the other hand, polyimide micromachining technology only allows for a maximum platin um electrode thick­ness of 1 Ilm, whi le in sil icone spiral cuffs platin um electrodes with a thick ness of 25 11m are used (Naples et al. , 1988). in order to increase the life of the implanted electrodes and because sil icone foils can not be micro­machined and the cuffs must be handmade. Polyi mide cuffs have lower volume and higher reproducibility than silicone cuffs, and can be micromachined in a variety of sizes and lengths suitable for implantation in small distal nerves, which could enhance the choices for selective st im ulation. Moreover, the chemically re-

versible charge injection limit for plat inum electrodes is about 40 llC/cm l fo r a geometric electrode surface area of 1.9 cm2 (Roblee and Rose, 1990), whereas 0.26- 4.15 I1C/cm2 were needed fo r maximal recruitment stimula­tion through the polyimide cuff electrode eva luated, ensuring reduced corrosion and a long life for the electrodes.

The use of loose fi tting cuffs may faci li tate the growt h of connective t issue inside the cuff and raise the impedance of the nerve - cuff interface. making it neccs­sal)' to apply larger stim uli over time (Krarup and Loeb. 1988; Naples et aI. , 1990; McCreery et al.. 1992; Loeb and Peck. 1996). We found no signi ficant changes between the mean threshold and maxima l recruitment stimulus intensity at the day of implantation and a fter 45 days. alt hough ch,mges occurred in single animals, with increased levels in five and decreased in four animals. Thi s is in agreement with the observations by Grill and Mortimer (1998) of considerable variability in the current req ui red to generate a particula r level of muscle activat ion fo r seven sessions during 35 weeks of follow up in the same animal. These authors att ri bu ted the tempora l variations to changes in the induced fi elds resulting from tissue encapsulation, changes in the elec­trode position relative to the motor nerve fi bers, and changes in the physiological properties of the ne uro­muscula r system incl ud ing degeneration and regenera­tion of the stimulated nerve fibers . However. we have not fo und a significant num ber of degenerating fi bers nor noticeable growth of connective tissue inside the cuff. The smoothness of the polyimide wall limits the adhesion of connective cells, preserving the conducting fl uid between the nerve and the electrode (Heiduschka and Thanos. 1998). Changes in threshold and maximal rccruitment stimulus found in individual ralS between both testing days could be explai ned by relative rota­tional movements of the nerve fascieles to the closest electrode surface until the fib rous tissue cover of the cufT fixes bot h structures.

In summary. we have developed and tested in vivo a flexible cuff electrode for peripheral nerve stim ulation or recording made of polyimide as in sulat ing carrier and three ring platin um elect rodes. The polyim ide cu ff electrode is mechanically harmless fo r the nerve and allows fo r stimulatiOn of mOtor and sensory large nerve fibers with in a wide margin of safety. Its reduced size opens the possibili ty of impla ntation around small dis­tal ncrves of larger mammals and conseq uently to achieve specific functional st imulation or recording at distal nerves. Future studies should be addressed to investiga te the abil ity of thcse nerve cuffs fo r longer term stimulation and to design new mullipolar cuffs useful for the selective stimulation of nerve fascicles lo improve the balance of alternating movements and to avoid muscle fatigue.

F.J. Rudrigue: el al. Journllloj N('uroJciell("" Un/u.Hfl 98 (:!()()()) 105- /18 "' Acknowledgements

The a uthors thank Ralf Keller for his work in thc cleanroom to fabricate the microdevices. This research has been supported by ESPRIT gra nl # 26322 (G RIP, Eel.

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