Ann. Medit. Burns Club - vol. 6 - n. 3 - September 1993

THE REGENERATION OF SKIN SENSITIVITY AFTER EXTENSIVE BURNS

Stella M., Magliacani G., Calcagni M., Panzica G.C., Ramieri G.

Divisione Chirurgia Plastica e Centro Grandi Ustionati, Ospedale C.T.O., Turin, Italy


SUMMARY. This work provides histological confirmation of superficial sensitive structures following scarring, and highlights the presence, in grafts, of fibres and superficial receptors. Fifteen patients were selected for the study, in which standard aesthesiological tests were used, integrated with the Weber and the Dellon test. The anti-protein gene product 9.5 was used to evidence the presence of nerve structures in normal skin, dermo-epidermic grafts and scars. The relative distribution of the structures regenerated in various anatomical regions is almost equal to normal, quantitatively speaking, even if the absolute number may be less.

Introduction

Extensive burns affect the nervous system at all levels. Lesions of the central nervous system, although they may jeopardize the patient's life, will not be considered here. However, in the peripheral nervous system, the lesion may occur in various forms, in all cases accompanied by degeneration and regeneration.
In the rehabilitation phase extensively burned patients frequently complain not only of their scarring but also of poor discriminative sensitivity, dysaesthesia and/or paraesthesia accompanied by peripheral circulatory deficit and dyshydrosis. The localization of the lesions responsible for these symptoms can vary considerably, creating a variety of physiopathological situations when a main nerve trunk, the peripheral plexus or the receptors are involved. The nerve may be damaged directly, as in the case of electrocution or full-thickness burns, when the nerve is subcutaneous (e.g. the sural). The surface plexus is involved when the thermal trauma affects the dermis (second-degree deep and thirddegree burns). As for the receptors, these may not only be directly damaged by the burn but they may also be affected by scarring processes, which alter their functional characteristics.
The surgeon must therefore carefully analyse the methods for the recovery of sensitivity and evaluate its qualities.

Materials and methods

For this study 15 patients were selected with dermo-epidermic grafts of at least 6 months' standing, together with 8 patients with spontaneous burn scarring sequelae. These were studied aesthesiologically by standard tests integrated with the Weber test and the Dellon test. Fourteen patients subjected to reconstructive plastic surgery for other pathologies involving the sacrifice of healthy skin acted as controls for the study.
Skin biopsies were performed and fixed in Zamboni liquid (4% paraformaldehyde and 0.2% picric acid in 0. IM phosphate buffer) for 12 h at 4 'C, washed in phosphate buffer (PBS) and 15% saccharose for 3 days, and then frozen.
The next step was the preparation of 15-30 W sections processed according to Sternberger's peroxidase-antiperoxidase method (1979). After deactivation of the endogenous peroxidases the sections were incubated overnight with anti-protein gene product 9.5 serum (PGP 9.5, Ultraclone, Cambridge, UK), diluted 1:3200 in PBS. Anti-IgG and PAP complex (Dako) were used (1:50) in PBS for I h at room temperature. The peroxidase reaction was developed in TRIS 0.05M buffer containing 0.02% diaminobenzidine and 0.01% (H202) per 10 M3. The immunocytochernical controls were performed: i) by replacing the primary antiserum with normal nonimmune rabbit serum, and ii) by omitting the antirabbit IgG or the PAP complex.

Results

Normal skin. A large number of nerve structures are evidenced by means of the anti-PGP 9.5 antibody in normal skin. It is possibile to observe a rich subpapillary plexus from which fibres branch out, cross the dermo-epidermic junction and penetrate the epidermis, where their route becomes variable.
Other fibres, at papillary level, form capsulate structures such as Merkel's complexes, Meissner's corpuscles, or simple ball-shaped complexes. Occasionally these structures are in pairs, innervated by ramifications of the same fibre.

Immunoreactive structures were also observed around vases, glands and hair bulbs.

Dermo-epidermic grafts. Examination of grafts of at least 6 months' standing showed a well-established subcutaneous plexus with fasciae running among the dermic papillae in a direction parallel to the dermoepidermic junction. From this plexus varicose fibres run straight into the epidermis.

Two types of receptors were observed:

1) free dermic endings, and
2) free intra-epithelial endings.

The latter, less numerous than the dermic receptors, were situated among the keratinocytes as far as the most superficial strata. In rare cases, immunoreactive structures were observed around the vessels.  Capsulate structures were not observed at all.
Scars. The anti-PGP 9.5 antiserum showed in scars the presence, below the dermic papillae, of a plexus with fasciae running parallel to the surface, from which varicose fibres branch off, giving rise to structures of variable complexity; other fibres cross the dermo-epidermic junction and form free endings in the epithelium.

Three types of receptors were observed:

1) free intraepithelial endings,
2) free dermic endings, and
3) Merkel's complexes.

The free endings were located among the keratinocytes as far as the most superficial layers. The Merkel complexes consisted of groups of round and ovoid cells in contact with specialized expansions of a single nerve fibre. The complexes were mostly situated in the basal stratum of the epidermis. No Meissner corpuscles were observed in any case. Immunoreactive structures were also observed around the vessels.

Discussion

Classical histological techniques (in particular argentic impregnation) made it possible to carry out the first studies on the nervous system, but they are unspecific and repetitive and unable to resolve certain doubts on the morphology and classification of fibres and receptors. In recent years technical advances in chromatography and electrophoresis have permitted the isolation and study of increasingly purer protein fractions, including the protein gene products. Used as an immunohistochernical marker, the PGP 9.5 has proved to be particularly useful for the study of the peripheral nervous system, both in the normal state (Wang, 1990) and when regenerating (Stella, 1990).
Histologically, it is possible to observe that the subpapillary plexus is always well represented in scars and grafts, even though its density does not reach that in normal skin.
Unlike other authors (Dalsgaard, 1989; Wang, 1990) we found some superficial structures, i.e. dermic and intraepithelial fibres.
With regard to the receptors we noted the constant presence of free endings both in the dermis and in the epithelium. There were no capsulated receptors either in the grafts or in the scars, but in the latter we observed some rare Merkel's complexes.

Conclusions

Classical aesthesiology recognized the recovery of sensitivity in the scar, often characterized by dysaesthesia and/or paraesthesia. The present work provides histological confirmation of the presence of superficial sensitive structures.
Regarding grafts, the clinical data to be found in the literature indicated poor recovery of sensitivity, with a protective component described as being like "feeling through a glove", while the histological data suggested the total absence of superficial structures (Dalsgaard, 1989; Wang, 1990). The present work highlights the presence, in grafts, of fibres and superficial receptors, even if of lower density than in normal skin. Quantitatively speaking, the relative distribution of the structures regenerated in the various anatomical regions is almost equal to normal, although the absolute number may be considerably less. A study is currently in progress aimed at the correlation of the clinical data and experimental observations.

RESUME. Cette étude offre une confirmation histologique de l'existence de structures sensitives superficielles à la suite de la cicatrisation, et met en lumière la présence, dans les greffes, de fibres et de recepteurs superficiels. Quinze patients ont partécipé à cette expérience, dans laquelle les auteurs ont utilisé les tests normaux esthésiologiques plus le test de Weber et celui de Dellon. L'anti-produit génétique protéique 9,5 a été utilisé pour manifester la présence de structures nerveuses dans la peau normale, les greffes dermoépidermiques et les cicatrices. La distribution relative des structures régénérées dans les diverses régions anatomiques est à peu près égale à l'état normal, du point de vue quantitatif, même si le numéro absolu peut être inférieur.


BIBLIOGRAPHY

  1. DaIsgaard C. L, Rydh Xl., Haegerstrand A.: Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies. Histochemistry, 92: 385, 1989.
  2. Stella M., Rarnieri G., Anselmetti G. C., Baracchi F_Caleagni M., Panzica G. C., Teich-Alasia S.: Dimostrazione di fibre nervose rigeneranti nella cute cicatriziale c trapiantata mediante un anticorpo contro la PGP 9.5. Riv. It.Chir. Plast., 22: 125, 1990.
  3. Sternberger L. X: The unlabelled antibody peroxidaseantiperoxidase (PAP) method. In "Immunocytochemistry", Sternberger L. A. (ed.), J. Whiley & Sons, New York, 104, 1979.
  4. Wang L., Hilliges M., Wiegleb-Edstrom D., Johansson 0.: Protein Gene Product 9.5 - immunoreactive nerve fibers and cells in human skin. Cell Tissue Res., 261: 25, 1990.



 

Contact Us
mbcpa@medbc.com