Stimulation of Trigeminal Afferents Improves Motor Recovery

After Facial Nerve Injury Functional, Electrophysiological

and Morphological Proofs

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In Memoriam

In memory of our distinguished colleague and friend Prof. Dr. Jürgen Koebke
(Department of Anatomy, University Hospital of Cologne, Germany) who died
suddenly on February 23, 2012. His father-like affection to students, sound
knowledge in anatomy and brilliant teaching will remain in the hearts of
thousands physicians and dentists for ever. Jürgen Koebke’s endless enthusiasm
for biomechanical and surgical research inspired hundreds of anatomy teachers,
abdominal and orthopedic surgeons, and traumatologists worldwide for decades.

v
.
Abstract

Recovery of mimic function after facial nerve transection is poor: the successful
regrowth of axotomized motoneurons to their targets is compromised by (1) poor
axonal navigation and excessive collateral branching, (2) abnormal exchange of
nerve impulses between adjacent regrowing axons, and (3) insufficient synaptic
input to facial motoneurons. As a result, axotomized motoneurons get hyperexcit-
able and unable to discharge. Since improvement of growth cone navigation and
reduction of the ephaptic cross talk between axons turn out be very difficult, we
concentrated our efforts on the third detrimental component and proposed that an
intensification of the trigeminal input to axotomized electrophysiologically silent
facial motoneurons might improve specificity of reinnervation. To test our hypoth-
esis we compared behavioral, electrophysiological, and morphological parameters
after single reconstructive surgery on the facial nerve (or its buccal branch) with
those obtained after identical facial nerve surgery but combined with direct or
indirect stimulation of the ipsilateral infraorbital (ION) nerve. We found that in all
cases, trigeminal stimulation was beneficial for the outcome by improving the
quality of target reinnervation and recovery of vibrissal motor performance.

vii
.
Acknowledgments

This work has been supported by the Jean Uhrmacher-Foundation, the Imhoff-
Foundation, and the Köln Fortune Programm. Special thanks to our colleagues
and friends Prof. Dr. Athanasia Alvanou, Prof. Dr. Sarah Dunlop, Dr. Maria
Grosheva, Dr. Marcin Ceynowa, Prof. Dr. Orlando Guntinas-Lichius, Dr. Peter
Igelmund, Privatdozent Dr. Andrey Irintchev, Prof. Dr. Katerina Kaidoglou,
Dr. Daniel Merkel, Dr. Anastas Popratiloff, Prof. Dr. Nektarios Sinis, and
Prof. Dr. Michael Streppel.

ix
.
Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1 First Major Set: Mild Indirect Stimulation of the Trigeminal Afferents
After Combined Surgery on the Infraorbital and Facial Nerves
by Removal (Clipping) of the Contralateral Vibrissal Hairs . . . . . . . . . . . . . . . . . . . . . 3
2.1.1 Animal Groups and Overview of the Specific Methods Used
in the First Experimental Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1.2 Combined Nerve Surgery (FFA þ ION-S) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1.3 Increased Ipsilateral Vibrissal Use (Vibrissal Stimulation, VS)
After Combined Surgery in Group 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.1.4 Manual Stimulation of Vibrissal Muscles After Combined Surgery
in Groups 3 and 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.1.5 Observations on Whisking Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.1.6 Analysis of Vibrissae Motor Performance During Exploration . . . . . . . . . . . 9
2.1.7 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.1.8 Analysis of Target Muscle Reinnervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.1.9 Synaptic Input to the Facial Motoneurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.1.10 Number of Retrogradely Labeled Trigeminal Ganglion Cells . . . . . . . . . . 16
2.1.11 Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.2 Second Major Set: Intensive Indirect Stimulation of the Trigeminal
Afferents After Facial Nerve Surgery by Excision of the Contralateral
Infraorbital Nerve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.2.1 Experiments to Determine the Degree of Collateral Axonal
Branching by Application of Fluorescent Dyes on the Transected
Superior and Inferior Buccolabial Rami of the Buccal Facial
Branch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.2.2 Experiments to Determine the Accuracy of Reinnervation
by Means of Intramuscular Injections of Fluorescent Dyes . . . . . . . . . . . . . 23
2.3 Third Major Set: Direct Stimulation of the Trigeminal and Facial
Nerves After Facial Nerve Surgery by Massage of the Vibrissal Muscles . . . . . . 28
2.3.1 Animal Groups and Overview of Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.3.2 Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.3.3 Standard Housing/Enriched Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.3.4 Mechanical Stimulation of the Vibrissal Muscles . . . . . . . . . . . . . . . . . . . . . . . . 34
2.3.5 Handling of the Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.3.6 Analysis of Vibrissae Motor Performance During Exploration . . . . . . . . . 34
2.3.7 Analysis of the Synaptic Input to the Facial Motoneurons . . . . . . . . . . . . . . 34

xi
xii Contents

2.3.8 Estimation of Axonal Branching by Triple Retrograde Labeling . . . . . . . 35

2.3.9 Analysis of Target Muscle Reinnervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2.3.10 Statistical Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2.4 Fourth Major Set: Direct Stimulation of the Trigeminal and Facial
Nerves After Facial Nerve Surgery by Application of Electric Current
to the Vibrissal Muscles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.4.1 Animal Groups and Overview of Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.4.2 Surgical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.4.3 Electrical Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.4.4 Analysis of Vibrissal Motor Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.4.5 Estimation of Axonal Branching by Triple Retrograde Labeling . . . . . . . 42
2.4.6 Analysis of Target Muscle Reinnervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.4.7 Statistical Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.1 Mild Indirect Stimulation of the Trigeminal Afferents After Combined
Surgery on the Infraorbital and Facial Nerves by Removal of the
Contralateral Vibrissal Hairs Improves Vibrissal Function . . . . . . . . . . . . . . . . . . . . . 43
3.1.1 Observations on Restoration of Vibrissal Whisking . . . . . . . . . . . . . . . . . . . . . 43
3.1.2 All Three Interventions (Sensory, Mechanical, and Sensory +
Mechanical Stimulation) Improved Vibrissal Function After
Combined Facial and Infraorbital Nerve Injury . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.1.3 For All Treatments (Sensory, Mechanical, and Sensory +
Mechanical Stimulation) Functional Outcome Correlates with
Quality of Target Muscle Reinnervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.1.4 Numbers of Synaptophysin-Positive Axon Boutons in the Facial
Nucleus Are Unaffected, Regardless of the Treatment . . . . . . . . . . . . . . . . . . . 46
3.1.5 No Neuronal Loss in the Trigeminal Ganglion After ION Lesion . . . . . . 47
3.2 Intensive Indirect Stimulation of the Trigeminal Afferents by Excision
of the Contralateral ION Attenuates the Degree of Collateral Axonal
Branching and Improves the Accuracy of Muscle Reinnervation . . . . . . . . . . . . . . 48
3.2.1 Reduced Degree of Collateral Axonal Branching as Determined
by Application of Two Fluorescent Dyes on the Transected Superior
and Inferior Buccolabial Rami of the Buccal Facial Branch . . . . . . . . . . . . . 48
3.2.2 Improved Accuracy of Reinnervation as Established by Means of
Intramuscular Injections of Fluorescent Dyes and Electrophysiological
Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.3 Direct Stimulation of the Trigeminal and Facial Nerves by Massage
of the Vibrissal Muscles Improves the Quality of Target Reinnervation
and Promotes Full Recovery of Whisking Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.3.1 Analysis of Vibrissae Motor Performance During Exploration . . . . . . . . . 60
3.3.2 Manual Stimulation Counteracts Posttraumatic Loss of
Synaptophysin-Positive Axon Terminals in the Facial Nucleus . . . . . . . . . 62
3.3.3 Degree of Collateral Axonal Branching Remains Elevated
Regardless of Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3.3.4 Mechanical Stimulation Reduces the Degree of Motor End Plate
Polyinnervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.3.5 Manually Stimulated Recovery of Motor Function After Facial
Nerve Injury Requires Intact Sensory Input . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Contents xiii

3.4 Direct Stimulation of the Trigeminal and Facial Nerves by Electric

Current to the Vibrissal Muscles Fails to Improve Quality of Target
Reinnervation and Does Not Promote Recovery of Vibrissal Function . . . . . . . . 65
3.4.1 Electrical Stimulation of the Vibrissal Muscles Does Not Promote
Recovery of Whisking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.4.2 A High Degree of Collateral Axonal Branching Occurs Regardless
of ES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.4.3 ES Does Not Reduce Polyinnervation of the Motor End Plates . . . . . . . . . 66
3.4.4 ES Reduces the Number of Motor End Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.1 Mild Indirect Stimulation of the Trigeminal Afferents by Removal of the
Contralateral Vibrissal Hairs Has a Beneficial Effect on Motor Recovery . . . . . 69
4.1.1 Importance of Sensory Fiber Regeneration for Motor
Axonal Regrowth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.1.2 Influence of Synaptic Coverage on Axonal Regrowth and Quality
of Target Reinnervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
4.1.3 Clinical Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.2 Beneficial Effect of the Intensive Indirect Stimulation of the Trigeminal
Afferents by Excision of the Contralateral Infraorbital Nerve . . . . . . . . . . . . . . . . . . 72
4.2.1 Removal of the Contralateral Trigeminal (ION) Input Attenuates
the Degree of Collateral Axonal Branching Within the Transected
Buccal Branch of the Facial Nerve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
4.2.2 Observations on the Recovering Vibrissal Function . . . . . . . . . . . . . . . . . . . . . 74
4.2.3 Removal of the Contralateral Trigeminal (ION) Input Improves
Quality of Whisker Pad Musculature Reinnervation . . . . . . . . . . . . . . . . . . . . . 76
4.3 Complete Recovery of Motor Function After Direct Stimulation of the
Trigeminal and Facial Nerves by Massage of the Vibrissal Muscles . . . . . . . . . . . 79
4.3.1 Methodological Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.3.2 Importance of the Stimulation Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.3.3 Possible Mechanisms of the Beneficial Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.3.4 Adverse Effect of Trigeminal Nerve Ablation on Functional
Recovery After FFA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.3.5 The Effect of Manual Stimulation Depends on the Integrity of the
Trigeminal Sensory System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.4 Deleterious Effect of the Direct Stimulation of the Trigeminal and Facial
Nerves by Application of Electric Current to the Vibrissal Muscles . . . . . . . . . . . 86
4.4.1 Rationale to Use Electrical Stimulation for Treatment
of Denervated Muscles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.4.2 Effect of Electrical Stimulation on the Quality of Muscle
Reinnervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

.
List of Abbreviations

AchE Acetylcholine esterase

BBA Buccal–buccal anastomosis, that is, transection and end-to-end
suture of the buccal branch of the facial nerve (enables regrowth
of the buccal branch of the facial nerve)
BSA Bovine serum albumin
CLSM Confocal laser scanning microscopy
CMAP Compound muscle action potential
Contra-ION-ex Excision of the contralateral infraorbital nerve
DiI 1,10 -Dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine per-
chlorate (red fluorescent dye)
DMSO Dimethyl sulfoxide
DPO Days postoperation
EE Enriched environment
ES Electrical stimulation
FB Fast Blue (blue fluorescent dye)
FFA Facial–facial anastomosis, that is, transection and end-to-end
suture of the facial nerve (enables regrowth of the facial nerve)
FG FluoroGold (yellow fluorescent dye)
HRP Horseradish peroxidase (retrograde neuronal label)
ION Infraorbital nerve
ION-ex Excision of the infraorbital nerve (causing degeneration of ION)
ION-S Transection and suture of the infraorbital nerve (enables
regrowth of ION)
Ipsi-ION-ex Excision of the ipsilateral infraorbital nerve
LLS Levator labii superioris muscle
MS Mechanical stimulation
NSE Neuron-specific enolase
NSS Normal sheep serum
SS Sham stimulation
TBS Tris-buffered saline
VS Vibrissal stimulation
WFM Widefield microscopy

xv
.
Chapter 1
Introduction

The facial nerve is the most frequently affected nerve in head and neck trauma.
Apart from traffic accident injuries (brainstem hemorrhage, temporal bone
fractures, or lacerations of the face), most of the facial nerve lesions are postopera-
tive (removal of cerebellopontine angle tumors, acoustic neuroma surgery, or
parotid resections because of malignancy). Despite the use of all available micro-
surgical techniques for repair of transected nerves, the recovery of facial tone,
voluntary movement, and emotional expression of the face remains poor (Anonsen
et al. 1986; Ferreira et al. 1994; Vaughan and Richardson 1993).
The inevitable occurrence of a “post-surgery paralytic syndrome” including
abnormally associated movements and altered blink reflexes (Kimura et al. 1975;
Bento and Miniti 1993) has been attributed to (1) “misdirected” reinnervation of the
facial muscles (Montserrat and Benito 1988; Sumner 1990), (2) trans-axonal
exchange of abnormally intensive nerve impulses between axons from adjacent
fascicles (Sadjadpour 1975), and (3) alterations in synaptic input to facial
motoneurons (Bratzlavsky and van der Eecken 1977; Graeber et al. 1993; Moran
and Neely 1996).
A key issue in these explanations, which do not exclude each other, is the
abnormal activity pattern of the axotomized facial motoneurons. On the one side,
the increase in resting potential and existence of still functioning axodendritic
synapses (Lux and Schubert 1975; Sumner and Watson 1971) renders them hyper-
excitable (Eccles et al. 1958; Ferguson 1978). On the other side, the decreased
synthesis of transmitter-related compounds (Lieberman 1971) and reduced
axosomatic synaptic input (Blinzinger and Kreutzberg 1968) render the axotomized
facial motoneurons less excitable upon afferent stimulation and unable to discharge
(Titmus and Faber 1990).
In our work over the last 2 decades we hypothesized that the abnormal activity,
that is, the axotomy-caused “silence” of the facial motoneurons, could be improved
by alterations in the input from the trigeminal sensory nucleus. This hypothesis
gains support from anatomical, electrophysiological, and clinical data showing the
involvement of the trigeminal system in generation of facial muscle responses and
blink reflexes (Moller and Jannetta 1986; Valls-Sole and Tolosa 1989).

E. Skouras et al., Stimulation of Trigeminal Afferents Improves Motor Recovery After 1

Facial Nerve Injury, Advances in Anatomy, Embryology and Cell Biology 213,
DOI 10.1007/978-3-642-33311-8_1, # Springer-Verlag Berlin Heidelberg 2013
2 1 Introduction

To test our hypothesis we compared behavioral, electrophysiological, and mor-

phological parameters after single reconstructive surgery on the facial nerve (or its
buccal branch) with those obtained after identical facial/buccal nerve surgery but
combined with direct or indirect stimulation of the trigeminal (infraorbital, ION)
nerve. We found that in all cases, trigeminal stimulation was beneficial for the
clinical outcome by improving the quality of target reinnervation and recovery of
vibrissal motor performance.
Chapter 2
Materials and Methods

Four major experimental sets have been performed using 413 rats to study the
effect(s) of:
1. Mild indirect stimulation of the trigeminal afferents (by clipping of the contra-
lateral vibrissal hairs)
2. Intensive indirect stimulation of the trigeminal afferents (by excision of the
contralateral infraorbital nerve)
3. Mild direct stimulation of the vibrissal muscles (by massage)
4. Intensive direct stimulation of the vibrissal muscles (by electric current)
All animals were young adult (175–200 g) female Wistar rats (strain HsdCpb:
WU, Harlan Winkelmann, Borchen, Germany). They were fed standard laboratory
food (Ssniff, Soest, Germany), provided with tap water ad libitum, and kept on a
12-h light–dark cycle. Experiments were conducted in accordance with the German
law on the protection of animals; procedures were approved by the local animal
care committee. We used only female rats because testosterone has been shown to
beneficially affect peripheral nerve regeneration (Yu and Yu 1983).

2.1 First Major Set: Mild Indirect Stimulation

of the Trigeminal Afferents After Combined Surgery
on the Infraorbital and Facial Nerves by Removal
(Clipping) of the Contralateral Vibrissal Hairs

2.1.1 Animal Groups and Overview of the Specific Methods Used

in the First Experimental Set

Forty-eight rats were randomly divided into four groups (Table 2.1).
Each group consisted of 12 rats that were subjected to unilateral combined injury
(transection and suture, i.e., anastomosis) of the right facial (FFA) and infraorbital

E. Skouras et al., Stimulation of Trigeminal Afferents Improves Motor Recovery After 3

Facial Nerve Injury, Advances in Anatomy, Embryology and Cell Biology 213,
DOI 10.1007/978-3-642-33311-8_2, # Springer-Verlag Berlin Heidelberg 2013
4 2 Materials and Methods

Table 2.1 Experimental design chart depicting animal grouping and procedures
Video-based Number of
motion Reinnervation retrogradely
analysis pattern of motor Synaptic labeled
of vibrissae end plates in input to the trigeminal
motor m. levator labii facial ganglion
Group of animals performance superioris motoneurons cells
1. FFA þ ION-S only 12 6 6 6
2. FFA þ ION-S þ VS 12 6 6 6
3. FFA þ ION-S þ MS 12 6 6 6
4. FFA þ ION-S þ VS þ MS 12 6 6 6
FFA and ION-S indicate transection and end-to-end suture of the facial and infraorbital branch
of the trigeminal nerves, respectively. VS ¼ trimming of contralateral vibrissae to maximize
ipsilateral vibrissal use for 4 months; MS ¼ manual stimulation for 4 months; VS/MS indicates
2 months of VS followed by 2 months of MS. All 12 animals in each group underwent video-based
motion analysis of the vibrissae motor performance. Thereafter, half of the animals were used to
study the reinnervation pattern of m. levator labii superioris. The other six rats in each group were
used for retrograde labeling of the facial and trigeminal neurons in the brainstem

(ION-Suture, ION-S) nerves. Rats from group 1 underwent surgery, but received no
postoperative therapy (FFA þ ION-S-only), whereas rats from groups 2–4 were
used to assess the efficacy of three treatment paradigms:
1. Removal of the contralateral vibrissae to ensure a maximal use of the ipsilateral
ones (vibrissal stimulation; FFA þ ION-S þ VS; group 2)
2. Manual stimulation of the ipsilateral vibrissal muscles (FFA þ ION-S þ MS;
group 3)
3. Vibrissal stimulation followed by manual stimulation (FFA þ ION-S þ VS
þ MS; group 4)
No intact controls were included in this major experimental set because our aim
was not to compare functional and morphological parameters between surgically
treated and intact animals: It is well known that after peripheral nerve injury,
recovery of function is poor and never reaches levels comparable to those in intact
animals.
Four months after combined surgery, all rats were videotaped to determine
vibrissal motor performance during explorative whisking using motion analysis
system (Table 2.2).
Thereafter, one half of the animals in each group were used to determine the
proportion of mono- and polyinnervated motor end plates (Table 2.3) in the
ipsilateral levator labii superioris muscle by means of immunocytochemistry for
neuronal class III b-tubulin and histochemistry with a-bungarotoxin (see below).
The animals from the second half were used to establish changes in the synaptic
input to the facial motoneurons that projected to the whisker pad muscles (identified
by retrograde labeling with FB, Table 2.4) and to document eventual changes in the
number of those trigeminal ganglion cells sending their dendrites into the
infraorbital nerve.