Sensibility and cutaneous reinnervation in free flaps

Sensibility and sensory reinnervation were investigated in 19 free flaps, predominantly located on the lower extremities, between 2 months and 3 years after flap transfer. All patients showed deep pressure sensibility. In 10 of the patients, primarily those examined late after surgery, a heat pain threshold was obtained at about 50 degrees C. None of the patients had superficial sensibility of any other modality. No neurofilament-positive sensory nerve fibers were observed in the dermis or epidermis. In one patient nerve fibers were detected in the subcutaneous tissue. It is concluded that patients will have deep pressure sensibility of the flap area even early after the operation and that most patients will develop a heat pain sensitivity, probably due to subcutaneous reinnervation.     

RT97: a marker for capsaicin-insensitive sensory endings in the rat skin

The mouse monoclonal antibody RT97, which recognises the 200-kDa neurofilament subunit in its phosphorylated form, selectively labels the somata of sensory A-fibres (large light cells) in the dorsal root ganglion of the rat. We have tested the hypothesis that this antibody also visualises large diameter sensory fibres and their end structures in peripheral tissue, in particular in the skin. RT97 immunoreactivity is found in endings that are known to be served by myelinated afferent fibres, including Meissner-like endings, Merkel discs, hair follicle receptors, Pacinian corpuscles and free nerve endings. RT97 immunoreactivity has not, however, been observed in endings of presumably unmyelinated sensory fibres (intraepidermal fibres immunoreactive for substance P and calcitonin gene-related peptide) or in sympathetic fibres innervating sweat glands and blood vessels. In addition, neither systemic (100–150 mg/kg as adults) nor perineural capsaicin pre-treatment affects RT97 immunoreactivity in the skin. The data indicate that RT97 is a useful marker in the study of the capsaicin-insensitive sensory innervation of the skin and possibly other peripheral organs.     

Laser-evoked brain potentials in patients with dissociated loss of pain and temperature sensibility

Brief heat stimuli, elicited by a CO₂ laser (10.6 μm wave length), activate the most superficial cutaneous nerve terminals of the thin myelinated Aδ and unmyelinated C fibres which mediate heat and pain sensations. This paper investigates late cerebral potentials (SEPs) in response to laser pulses in comparison with those to conventional electrical stimulation in 18 patients with a dissociated sensory deficit (intact mechanosensibility and disturbed temperature and pain sensation). Patients were stimulated in the most disturbed limb (affected area) and in a corresponding control area.In all 18 patients the SEPs elicited by laser stimuli were able to identify the body site with heaviest disturbances in pain and thermosensibility: the SEPs from the affected area were reduced or delayed, compared to the control area. In contrast, no alterations in SEPs could be observed after conventional electrical nerve stimulation, in agreement with the normal mechanosensibility. However, the degree of SEP modulation in response to cutaneous heat stimuli did not correspond to the severity of the subjectively reported sensory deficit. Highest correlations between sensory deficits and abnormal SEPs were found in all those patients in whom computer tomography or MR imaging documented a localized destructive process in the CNS. All patients with the smallest SEP modulations despite a considerable sensory deficit had an inflammatory aetiology. Preliminary criteria to define a laser-evoked SEP as pathological are discussed.     

Free flap reconstruction of the sole of the foot with or without sensory nerve coaptation

The authors present a retrospective study on major plantar foot reconstruction to evaluate the role of the free fasciocutaneous flap and the importance of sensory nerve reconstruction in improving long-term results. Between 1995 and 1999, 20 patients with major defects of the sole of the foot underwent free forearm flap reconstruction performed by the senior author (F.S.). Sensory nerve reconstruction was added to this technique in 1997. The age and sex of the patients and the cause, location, and dimensions of their defects were recorded. The patients were clinically and neurophysiologically evaluated at 3, 6, and 12 months after the procedure for the following parameters: flap contour, flap stability, load capacity, walking ability, touch sensation, pain sensation, static two-point discrimination, and thermal sensibility. Dermatomic somatosensory-evoked potentials were also tested at 12 months. Follow-up ranged from 1 to 5 years. Patients were divided into two groups according to sensory nerve reconstruction. Group A consisted of 11 patients with nerve repair, and group B consisted of nine patients without nerve repair. One patient from group A who had an idiopathic neuropathy was excluded from the study because of interference with the reinnervation process. Five more patients (three from group A and two from group B) were lost at follow-up and excluded from the study. The final sample size in each group was seven. Data from both groups were compared and statistically analyzed with the Mann-Whitney test and the Fisher exact test. Long-term results confirmed in all reconstructions long-lasting stability. During the first postoperative year, patients with sensory nerve reconstruction showed better sensibility. The statistical analyses confirmed significant differences between the two groups to be dependent upon surgical technique at 3 and 6 months. Two-point discrimination and dermatomic somatosensory-evoked potentials were recorded. After 12 months, flaps without surgical nerve repair showed progressive improvement of sensitive thresholds, achieving a good protective sensibility, similar to that of the other group, but these flaps never regained two-point discrimination or dermatomic somatosensory-evoked potentials.     

Sensory reinnervation in microsurgical reconstruction of the heel

Six sensory reinnervation techniques were carried out in 10 patients who underwent reconstruction of the weight-bearing surface of the heel by microsurgical free-tissue transfer. The techniques include the use of neurovascular island flaps, neurosensory flaps, sensory nerve grafts to skin flaps, coaptation of the sensory nerve to the motor nerve of the muscle flaps, direct sensory nerve transfer, and sensory nerve graft transfer. In all patients, some sensation developed, characterized by sensation to light touch, to dull objects, to pinprick, to pain, and to tickling. Three patients developed the ability to distinguish sharp from dull objects and the sensation of pain. The remaining seven had the sensation of touch to various mechanical stimuli. In nine patients, the sensation is located in the weight-bearing surface of the reconstructed heel. Five patients bear weight on the reconstructed surface at least 6 hours per day. Three participate actively in sports. Split-thickness skin graft-muscle flaps were more prone to breakdown than skin flaps. Full-thickness skin flaps appear necessary for the production of pain sensation and the more discriminating sensations. Preliminary results suggest a functional benefit after sensory reinnervation.     

Normal cutaneous sensibility of the face

Normal values for facial sensibility were determined in 36 healthy subjects. Sensation was evaluated using static and moving two-point discrimination and pressure and vibratory threshold in four major regions of the head and neck. Each region was directly related to the area innervated by a particular peripheral sensory nerve (supraorbital, infraorbital, inferior alveolar-mental, or the great auricular nerve). Cutaneous sensibility varied from region to region but was consistent from one normal individual to another and in the same subject on different days. Measurements of pressure and vibratory thresholds provided the most reliable, reproducible data. A relationship of these data to the types of sensory receptors in facial skin and vermilion is postulated.     

Ultrastructural examination of B-50(GAP-43) immunoreactivity in rat jejunal villi

Summary The lamina propria of rat jejunum is densely innervated with nerve fibres extending to the tips of the villi. A large number of these nerve fibres were previously shown to be B-50-immunoreactive at the light microscope level, whereas neurofilament immunoreactivity was found to be sparse in the mucosa. In this study we used immunoelectron microscopy to determine what proportion of nerve fibres in the lamina propria express B-50. Jejuna from male Lewis rats were immunolabelled for B-50 and neurofilament proteins. For electron microscopy, postembedding immunogold-silver techniques and LR White embedded tissues were used. Light microscopical immunostaining was performed by the streptavidin-biotin-peroxidase technique on deparaffinized tissue sections. We found that all ultrastructurally identifiable nerve profiles in jejunum were B-50 immunoreactive. Immunoelectron microscopy for neurofilament proteins failed to label fibres in the villi, whereas myelinated nerves in tongue sections processed in parallel (positive controls) were strongly neurofilament-protein-immunoreactive. The dominant B-50-positive and neurofilament-protein-negative phenotype supports the hypothesis of ongoing modelling or plasticity of intestinal mucosal nerves.     

Distribution of neurofilament-immunoreactive nerve fibers in human skin

Neurofilament immunoreactive nerve fibers were demonstrated in human skin using indirect immunohistochemical technique with antibodies to neurofilament polypeptides. Neurofilament-positive fibers were seen as free nerve endings in the epidermis and in dermal papilla, in Meissner's corpuscles and as fibers crossing in the dermis. Strongly fluorescent nerve fibers were also seen around hair follicles, sweat gland ducts and sometimes in relation to blood vessels. From the distribution pattern it was concluded that predominantly sensory nerve fibers were labelled and that this technique may be used to study reinnervation of cutaneous sensory nerves following traumatic injuries and surgical procedures.     

Influence of human skin injury on regeneration of sensory neurons

The regeneration of sensory nerve fibres is regulated by trophic factors released from their target tissue, particularly the basal epidermis, and matrix molecules. Means to modulate this response may be useful for the treatment of neuromas and painful hypertrophic scars and of sensory deficits in skin grafts and flaps. We have developed an in vitro model of sensory neuron regeneration on human skin in order to study the mechanisms of sensory dysfunction in pathological conditions. Adult rat sensory neurons were co-cultured with unfixed cryosections of normal or injured (crushed) human skin for 72 h. Neurons were immunostained for growth-associated protein-43 and the neurite lengths of neuronal cell bodies situated in various skin regions were measured. Two-way analysis of variance was performed. Neurites of sensory cell bodies on epidermis of normal skin were the shortest, with a mean +/- SEM of 75+/-10 micrometer, whereas those of cells on the dermo-epidermal junction were the longest, with a mean +/- SEM of 231+/-18 micrometer. Neurons on the dermo-epidermal junction of injured skin had significantly longer neurites than those on the same region of normal skin (mean +/- SEM = 289+/-21 micrometer). Regeneration of sensory neurons may be influenced by extracellular matrix molecules, matrix-binding growth factors and trophic factors. Altered substrate or trophic factors in injured skin may explain the increase of neurite lengths. This in vitro model may be useful for studying the molecular mechanisms of sensory recovery and the development of neuropathic pain following peripheral nerve injury.     

Distribution of neurofilament-immunoreactive nerve fibers in human skin

Summary Neurofilament immunoreactive nerve fibers were demonstrated in human skin using indirect immunohistochemical technique with antibodies to neurofilament polypeptides. Neurofilament-positive fibers were seen as free nerve endings in the epidermis and in dermal papilla, in Meissner's corpuscles and as fibers crousing in the dermis. Strongly fluorescent nerve fibers were also seen around hair follicles, sweat gland ducts and sometimes in relation to blood vessels. From the distribution pattern it was concluded that predominantly sensory nerve fibers were labelled and that this technique may be used to study reinnervation of cutaneous sensory nerved following tramatic injuries and surgical procedures.     

Combination of non-specific cholinesterase histochemistry and immunofluorescence staining for the study of the sensory innervation of skin and muscle

Summary In the present study we describe the application of the non-specific cholinesterase (nChE) histochemical method for the detection of encapsulated sensory nerve endings prior to immunofluorescence staining of the sensory nerve fibres. The nChE staining of Schwann-derived structures surrounding sensory terminals allowed us to identify unequivocally the sensory corpuscles in the skin and the muscle proprioceptors (muscle spindles and Golgi tendon organs) in longitudinal sections of muscle tissue. The nChE staining of sensory nerve endings and immunofluorescence-labelled nerve fibres and their terminals could be viewed and photographed in the same section using appropriate filters. Since nChE activity persists in terminal Schwann cells for a long time after loss of the sensory axons, this combined enzyme- and immunohistochemical approach is also useful for experimental studies involving denervation and re-innervation of sensory nerve endings.     

Allodynic skin in post-herpetic neuralgia: histological correlates

Most post-herpetic neuralgia (PHN) patients suffer from tactile allodynia (pain evoked by lightly touching the skin) and it is frequently the dominant clinical manifestation. The pathophysiology of tactile allodynia in PHN patients is poorly understood and this is one of the major limits to the development of appropriate therapies. Epidermal nerve fibres (ENFs) are free nerve endings of small-diameter A-delta and C primary afferents, which can easily be assessed by neurodiagnostic skin biopsy (NSB). The aim of this study was to establish the correlation between the residual epidermal innervation of the allodynic skin and the intensity of tactile allodynia in that area. Twenty-five patients (13 males and 12 females) with PHN were enrolled. Eighteen patients had PHN in the thoracic dermatome, four in the cervical, two in the trigeminal and one in the lumbar. The severity of allodynia evoked by a paintbrush was graded according to an eleven-point numerical scale. A skin biopsy was obtained from the maximal allodynia area and from the contralateral skin. Nerve fibres were labelled with indirect immunofluorescence. Results showed that epidermal innervation was lower in the allodynic skin than in the contralateral skin, although there was great variability among patients. There was no correlation between severity of allodynia and epidermal innervation of the PHN skin. In conclusion, the present study further indicates peripheral nervous system involvement in PHN but does not support a direct correlation between epidermal innervation changes and tactile allodynia.     

Use of the novel contact heat evoked potential stimulator (CHEPS) for the assessment of small fibre neuropathy: correlations with skin flare responses and intra-epidermal nerve fibre counts

Background  

The Contact Heat Evoked Potential Stimulator (CHEPS) rapidly stimulates cutaneous small nerve fibres, and resulting evoked potentials can be recorded from the scalp. We have studied patients with symptoms of sensory neuropathy and controls using CHEPS, and validated the findings using other objective measures of small nerve fibres i.e. the histamine-induced skin flare response and intra-epidermal fibres (IEF), and also quantitative sensory testing (QST), a subjective measure.     

Changes in fibre populations of the rat hairy skin following selective chemodenervation by capsaicin

Perineural application of capsaicin results in a selective and permanent reduction in the sensitivity to noxious chemical and heat stimuli and elimination of the neurogenic inflammatory response. The present quantitative immunohistochemical study has been undertaken to reveal the populations of cutaneous afferent nerves that are affected by perineural capsaicin treatment. Areas of intact and chemodenervated skin were determined with the aid of the vascular labelling technique. In sections taken from intact skin areas, staining with antibodies against protein gene product 9.5 revealed a rich epidermal innervation. Fibres immunoreactive for growth-associated protein 43 were also abundant; nerve fibres immunoreactive for substance P and calcitonin gene-related peptide were less numerous. Somatostatin- and RT97-immunoreactive fibres were seen only in the subepidermal layer. In sections taken from skin areas supplied by the sciatic nerve treated with capsaicin 3 days previously, the number of epidermal nerve fibres immunoreactive to protein gene product 9.5, growth-associated protein 43, substance P and calcitonin gene-related peptide was reduced by 90%, 95%, 97% and 66%, respectively. These changes persisted for at least 42 days. The findings reveal that the majority of epidermal axons are capsaicin-sensitive and comprise a chemically heterogeneous population. Reductions in cutaneous fibre populations following perineural capsaicin treatment may result from both the degeneration of sensory axons and the depletion of neuron-specific macromolecules. In addition, most cutaneous nociceptive axons may not use the major sensory neuropeptides substance P and calcitonin gene-related peptide as afferent neurotransmitters.     

Sensory restoration of the skin graft on a free muscle flap: experimental rabbit study.

Transplantation of a muscle flap with free skin graft for wound coverage is a common procedure in reconstructive microsurgery. However, the grafted skin has little or no sensation. Restoration of the sensibility of the grafted skin on the transferred muscle is critically important, especially in palmar hand, plantar foot, heel, and oral cavity reconstruction. The purpose of this study was to investigate the possibility of sensory restoration of the grafted skin on a trimmed muscle surface that has been sensory neurotized after sensory nerve-to-motor nerve transfer, using the rabbit gracilis muscle as an animal model. The ipsilateral saphenous nerve (sensory) was transferred to the motor nerve of the gracilis muscle for sensory neurotization. A 4 x 4-cm2 area of skin island over the midportion of the gracilis muscle was harvested as a full-thickness skin graft. The upper half of the gracilis muscle was then excised, becoming a rough surface. The harvested skin was reapplied on the trimmed rough surface of the muscle. After 6 months, retrograde and antegrade horseradish peroxidase labeling studies were performed through skin and muscle injection. The group with a free skin graft was compared with the group with an intact surface of the gracilis muscle. This study clearly shows that sensory nerves can regenerate and penetrate into the trimmed muscle surface and grow into the overlying grafted skin. However, if the muscle surface is intact as with the compared group, sensory reinnervation of the grafted skin is not possible.     

Immunohistochemical study of sensory nerve formations in human glabrous skin.

The sensory nerve formations (or corpuscles) of normal human glabrous skin from hand and fingers, obtained by punch biopsies, were studied by the streptavidin-biotin method using monoclonal antibodies directed against neurofilament protein (NFP), S-100 protein, glial fibrillary acidic protein (GFAP), cytokeratins, and vimentin. NFP immunoreactivity (IR) was observed in the central axons of most sensory formations, while S-100 protein IR was restricted to non-neuronal cells forming the so-called inner cells core or lamellar cells. Furthermore, vimentin IR was found in the same cells of Meissner's and glomerular corpuscles. None of the sensory nerve formations were stained for GFAP or keratin. The present results suggest that the main nature of the intermediate filaments of the non-neuronal cells of sensory nerve formations from human glabrous skin is represented by vimentin and not by GFAP. Thus, our findings suggest that lamellar and inner core cells of SNF are modified and specialized Schwann cells and not epithelial or perineurial derived cells.     

Human Skeletal Muscle Cells Synthesise a Neuronotrophic Factor Reactive with Spinal Neurons

Retrograde trophic influences originating in the skeletal musculature have been postulated to be involved in regulating survival and differentiation of embryonic motor neurons and reactive terminal sprouting of mature motor fibres. We have previously described the use of a quantitative immunoassay for neurofilament protein to bioassay in vitro the cell-type-specific neuronotrophic activity of nerve growth factor (NGF) on sensory ganglion neurons. In the present study, the effect of media conditioned by adult human muscle cells (MCM) on the in vitro development of chicken spinal neurons has been studied using a similar approach. Significant increases in neurofilament protein levels in 7-day chicken embryonic spinal cord cultures were found with doses of MCM protein as low as 0.4 microgram/ml, with a dose-response relationship yielding maximal and half-maximal effects at 4 and 1 microgram/ml, respectively. Maximal increases in neurofilament protein levels were associated with an approximate two-fold increase in neuronal cell survival. MCM also induced increases in choline acetyltransferase activity in chick spinal cord cultures. In both the absence and presence of NGF, MCM did not increase neurofilament protein expression in primary cultures of sensory neurons.     

Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies

Using antibodies to the neuronal cytoplasmic protein, protein gene product 9.5 (PGP 9.5) the cutaneous innervation in man was investigated. The distribution of PGP 9.5 immunoreactive nerve fibers was compared with the distribution of nerve fibers immunoreactive to neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y. PGP 9.5 immunoreactive nerve fibers were found in the epidermis, dermis, in Meissner's corpuscles, innervating Merkel cells, around blood vessels, sweat glands and hair follicles. Merkel cells were also PGP 9.5 positive. The labelled nerve fibers included sensory and autonomic fibers, visualizing the whole innervation of the human skin. The number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies than with any of the other markers included. Thus, PGP 9.5 antibodies may serve as a tool for investigations of cutaneous innervation, reinnervation and nerve regeneration in different clinical conditions.     

Ultrastructural studies in rheumatoid polyneuropathy

The biopsy material from the sural nerve of 8 patients suffering from rheumatoid arthritis and showing, with one exception, positive serological reactions, were studied by means of electron microscopy. All the patients were displaying sensory and sensomotor neuropathy. Besides hypermyelination the damage of Schwann cells, the destruction on unmyelinated fibres and the proliferation and degeneration of the vascular endothelium were observed. No inflammatory signs were seen around the peri- and endoneurial vessels.