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Long-term results of autologous submandibular gland transfer for the surgical treatment of severe keratoconjunctivitis sicca
Journal of Cranio-Maxillofacial Surgery (2008) 36, 227e233 Ó 2008 European Association for Cranio-Maxillofacial Surgery doi:10.1016/j.jcms.2007.08.010, available online at http://www.sciencedirect.com
Long-term results of autologous submandibular gland transfer for the surgical treatment of severe keratoconjunctivitis sicca Hans-Christian JACOBSEN1, Samer G. HAKIM1, Isabel LAUER2, Andreas DENDORFER3, Thilo WEDEL4, Peter SIEG1 Department of Maxillofacial Surgery (Chairman: Prof. Dr. Dr. P. Sieg), University of Luebeck, Germany; 2 Department of Radiology and Nuclear Medicine (Chairman: Prof. Dr. T. Helmberger), University of Luebeck, Germany; 3 Institute of Pharmacology and Toxicology (Chairman: Prof. Dr. P. Dominiak), University of Luebeck, Germany; 4 Institute of Anatomy (Chairman: Prof. Dr. J. Westermann), University of Luebeck, Germany
1
Aim: The aim of this study was to assess the long-term results of autologous submandibular gland transfer for surgical correction of severe keratoconjunctivitis sicca. Patients and methods: A survey was undertaken of 32 patients who had undergone submandibular gland transfer (42 glands) and by following up 11 patients (15 glands) for 5e10 years. Subjective benefit was evaluated as well as clinical findings at the ocular surface. The biochemical consistency of the secreted ‘‘saliva-tear’’ was analysed and compared with natural submandibular saliva of a matched control-group. The vitality and function of the transplants was tested by means of sialoscintigraphy. Immunohistochemical investigations were carried out in specimens of submandibular tissue, gained during reduction procedures of the transplants to correct secretory excess. Results: Patient evaluation and clinical assessment revealed a long-lasting subjective benefit in 2/3 of the patients and a stabilisation at the ocular surface in all cases. The secretion remained as highly concentrated submandibular saliva. Glandular vitality and function was shown scintgraphically. Immunohistochemical investigations revealed no progressive atrophy after transplantation, the ability of cell division remained intact and there was still neuronal tissue in all transplants, even several years after transfer. As all transplants responded well to parasympathomimetic drugs, this might be an indication of re-innervation of the gland. Ó 2008 European Association for CranioMaxillofacial Surgery
SUMMARY.
Keywords: submandibular gland transfer, keratoconjunctivitis sicca, long-term results, microvascular surgery, sialoscintigraphy
plantation of oral mucosal grafts into the conjunctival fold and/or obliteration of the lacrimal drainage pathways. A further therapeutic option was first suggested by Murube-del-Castillo in 1986, who described the microvascular autologous submandibular gland transfer for surgically correcting severe keratoconjunctivitis sicca. For complicated cases, this has been the common procedure in Luebeck for more than 10 years. This procedure requires extensive interdisciplinary coordination and is currently clinically established in only a few centres in Australia, China, the UK and Germany. Very few publications are available on the follow-up of these patients. They all describe the results within the first 2 years postoperatively (Murube-del-Castillo, 1986; Kumar et al., 1991; Sieg and Schirner, 1995; Geerling et al., 1998; Sieg et al., 2000; Yu et al., 2004). There remain several questions regarding the long-term follow-up of keratoconjunctivitis as well as the long-term stability of the function and morphology of the revascularised gland. As far as we know this paper is the first to report the long-term follow-up after submandibular gland transfer.
INTRODUCTION Dry eye syndrome is one of the most frequently occurring ophthalmological conditions. Its prevalence lies between 5 and 58% depending on selection of study groups and criteria recommended for diagnosis (Schaumberg et al., 2003; Moss et al., 2004). The development of keratoconjunctivitis sicca is proven to be associated with increasing age and female gender; alone in China about 30 million people suffer from this disorder (Yu et al., 2004). From a biological point of view, dry eye and the resulting keratoconjunctivitis is produced by the inadequate interrelation between lacrimal film and ocular surface epithelium, caused by quantitative and qualitative deficits in one or both of them. Without adequate treatment this chronic condition almost always produces permanent discomfort for the patient and is followed by a progressive destruction of the corneal surface leading to recurrent infections and visus restriction or even loss of the eye. Symptomatic treatment includes the application of tear substitutes first of all, surgical corrections such as tarsorrhaphy or im227
228 Journal of Cranio-Maxillofacial Surgery
PATIENTS AND METHODS Patients The aim was to revaluate patients who had undergone submandibular gland transfer more than 5 years ago. Out of 11 such patients, four underwent a bilateral procedure in two steps, so the total number of transferred glands was 15. The mean time between operation and evaluation was 6.4 + 0.8 years. The mean age was 53.4 + 19.4 years and eight of the 11 were female. The underlying pathology causing keratoconjunctivitis sicca was heterogeneous (Table l). Selective submandibular saliva was collected from a matched control-group of healthy patients in order to investigate the biochemical quality of the secretion and the response to pain sympathetic stimulation of the gland. Surgical technique The surgical procedure consisted of harvesting the submandibular gland with its supplying blood vessels and the excretory duct from a combined cervical and intra-oral approach (Fig. 1). The gland was transferred into a preformed pocket in the temporal muscle (Figs. 2 and 3). The blood vessels were anastomosed microsurgically to the temporal vein and artery (Fig. 4). The excretory duct was led subcutaneously to the lateral canthus and implanted into the upper lateral fornix of the conjunctiva. The key points of the procedure have already been described in detail (MacLeod et al., 1990; MacLeod and Robbins, 1992; Sieg and Schirner, 1995; Sieg et al., 2000; Yu et al., 2004). Methods Evaluation included a subjective assessment using a questionnaire, a clinical examination of the ocular surfaces and conjunctivae, a biochemical analysis of the ‘‘saliva-tears’’ in comparison with regular submandibular saliva collected from the matched control-group, and sialoscintigraphy for functional assessment of the gland. To gain more information about the structural changes in the transplants, specimens were examined from tissue taken during reduction procedures of transplanted glands. These procedures became necessary due to epiphora or persisting excessive secretion and were carried out 9e88 months (mean 26.5 months) after transplantation. Specimens from 11 cases were investigated immunohistochemically and compared with six specimens of nontransplanted submandibular tissue, removed during neck dissection procedures. Patient questionnaire and clinical evaluation The patients were sent a questionnaire including the question whether they would undergo the procedure again considering the present result. Further questions evaluated factors such as physical activity, warm temperature, and the effect of wind or chewing on the secretion as well as the changes of secretion during the daytime.
These questions had to be rated semi-quantitatively with values from 0 to 10 points. Clinical evaluation consisted of measuring the basal secretion using Schirmer’s test (Collins and Augustin, 1997). In order to evaluate the stimulation of secretion, a parasympathetic drug (0.2 mg Carbachol) was administered subcutaneously and Schirmer’s test was repeated after 10 min. The ocular surfaces were evaluated by slit-lamp examination after rose Bengal staining. These results were compared with the findings made prior to transplantation. Biochemical analysis The saliva-tears were collected before and after medical parasympathetic stimulation. They were examined for sodium, chloride, calcium phosphate and the whole protein content; and the osmolality was also evaluated. Specific proteins like kallikrein, peroxidase and secretory immunoglobulin A (slgA) were analysed to show the function of relevant cell-types in the transferred glands. Sialoscintigraphy Salivary gland scintigraphy was performed using a double head gamma camera (Picker/Marconi Prism 2000 S, Marconi Medical Systems, Cleveland, USA) with low energy ultra high resolution collimator. Directly after administering 50 MBq 99mTc-pertechnetate intravenously, dynamic scintigrams of the head (lateral views) were acquired over a period of 45e 50 min (1 frame/min, matrix 128 128, zoom factor 1.3). Salivary excretion was stimulated by subcutaneous administration of 0.2 mg Carbachol after 20 min. In order to analyse the scintigrams, regions of interest (ROIs) of the transplants were marked and time activity curves were generated (Figs. 9 and 10). Percentage tracer uptake before and after administration of Carbachol was calculated using an additional background ROI (Uptake before Carbachol Upbefore, Uptake after Carbachol Upafter). The salivary excretion fraction (EF) was quantified according to the following formula (Liem et al., 1996; Bohuslavizki et al., 1997): EFð%Þ ¼ ðUpbefore Upafter =Upbefore Þ 100. The EF of the parotid gland and, if possible, of the submandibular gland of the opposite side was calculated in the same way. Histology and immunohistochemistry The existing biopsy samples were cut at 2e6 mm and dewaxed. The amount of fat and fibrosis was measured (in %) in all sections after digital binary separation using the soft-imaging system ANALYSISÒ. Standardised immunohistochemical staining with monoclonal antibodies (DakoÔ, Glostrup, Denmark) was performed. The staining with alpha smooth muscle actin (ASMA) for the myofilaments in smooth muscle cells was an indicator for degenerative processes in salivary gland tissue (Hakim et al., 2002). Monoclonal Ki 67 antibodies (clone MIB 1) were applied to find proliferating cells in the (transferred) gland parenchyma. The reaction to protein gene product (PGP) 9.,5 and protein S-100 antibody showed the presence of neuronal ganglionic cells and nerve fibres, respectively. As chromogen a commercial
Long-term results of autologous submandibular gland transfer 229 Table l e Underlying causes of keratoconjunctivitis sicca Causes of keratoconjunctivitis sicca Patients (n ¼ 11) Traumatic loss of lacrimal gland Facial palsy nerve Benign cicatrical pemphigoid Lyell syndrome Congential aplasia of gland Idiopathic disease
1 2 1 2 1 4
Fig. 2 e Receptor area in the left temporal region. Fig. 1 e Harvested submandibular gland with supplying vessels and excretory duct.
kit with marking antibodies (ACE-KITÒ by ZymedÔ, Munich, Germany) was used. The analysis was performed at 100 magnification with a ZeissÒ graticule with 10 10 grids. RESULTS Patient questionnaire The results of the questionnaire showed that 53% of the patients were satisfied with the long-term result of the procedure, 13% were indifferent and 33% felt that the subjective outcome was not satisfactory when compared with the effort of treatment (Fig. 5). Fifty percentage of patients reported phases of hypo-secretion and 65% of hyper-secretion during the day, indicating that both inadequate conditions could occur in the same patient. During the day the secretion decreased by about 50% on average. Factors increasing secretion were warmth and physical activity (Fig. 6). Clinical evaluation The basal secretion of 4.5 ^ 4.9 mm mean as evaluated by the Schirmer’s test was increased significantly ( p ¼ 0.012) to 14.7 ^ 15.9 mm by stimulation with Carbachol (Fig. 7). There was whilst ophthalmologic examination a distinct reduction in the use of artificial tears in 30%
Fig. 3 e Prepared pocket with temporal vessels.
230 Journal of Cranio-Maxillofacial Surgery Factors influencing secretion 10 9 8 7 6 5 4 3 2 1 0 Warm temp.
Wind
Chewing
Physical activity
Fig. 6 e Factors influencing the secretion as taken from the questionnaire, rated semi-quantitatively with values from 0 to 10 points for surrounding temperature, wind, chewing and physical activity.
Fig. 4 e Transplanted gland with anastomosed vessels.
Fig. 7 e Schirmer test results (medians) from 15 transplants in mm, at rest and 10 min after stimulation with Carbachol.
of the cases revealed that the vision had not changed significantly after the surgical procedure. The cornea showed a significant improvement of lubrication in about 45% of patients.
significantly increased in the ‘‘saliva-tears’’ except for calcium and phosphate which showed a reduction. The changes of concentration on stimulation with Carbachol were similar to the changes in the secretion of the control-glands. The relatively increased concentrations of kallikrein, peroxidase and slgA as well as the changes following stimulation with Carbachol showed the specific secretory ability of acinar and ductal cells in the transplanted glands (Table 2). The essential differences between chemical composition of native submandibular saliva, the secretion of the transplanted gland and physiological tear fluid are listed in Table 3 for electrolytes, osmolality and total protein (Lutz, 1997).
Biochemical investigation
Sialoscintigraphy
Data from the biochemical analysis of the ‘‘saliva-tears’’ produced with and without stimulation were compared with the native saliva of the matched control-group. The concentrations of all investigated components were
Vitality and excretion capacity of transplanted glands could also be demonstrated by using sialoscintigraphy (Fig. 9). Excretory function was shown by stimulating the glands with Carbachol and calculating the salivary
Fig. 5 e Subjective benefit of treatment as taken from the questionnaire.
Long-term results of autologous submandibular gland transfer 231 Table 2 e Differences in concentration of salivary biochemical components between transplants and control-glands and changes following stimulation with Carbachol Biochemical concentrations
Sodium Chloride Calcium Phosphate Osmolality Total protein Kallikrein Peroxidase sIgA
Secretion of transplant compared with control
Changes in transplant secretion following stimulation with CarbacholÒ
+ + + + + + +
+ + ^
Fig. 8 e Time activity curve of tracer in the region of the 10 transplants, showing the uptake after technetium tracer injection and clearance after stimulation with Carbachol versus time.
Table 3 e Differences of chemical composition between normal tear fluid, physiological submandibular saliva and transplant secretion Chemical composition of lacrimal and salivary secretion
Sodium (mmol/1) Chloride (mmol/1) Calcium (mmol/1) Phosphate (mmol/1) Osmolality (mosmol/kg) Total protein (g/1)
Lacrimal fluid (Data from the literature)
Submandibular saliva (control group)
Transplant secretion (patients)
146* 128* 0.57* 0.07y 326* 6.69*
14 19.6 1.48 4.12 44.75 0.31
28.25 64 2.23 2.62 71.75 0.5
* Wissenschaftliche Tabellen Geigy (1977). y Lutz (1997).
production following stimulation. All transferred glands displayed a significant decrease of tracer uptake 5 min after injection of Carbachol (Fig. 8). The late scan 25 min after stimulation showed also the accumulation of tracer around the eye and in the mouth as a sign that the tracer was eliminated by secretion (Fig. 10). This proved again the response of parasympathomimetic neuroreceptors in the transplants. Histological findings Morphometry showed an average of 30% of fatty tissue and fibrosis in the transplants against 10% in the control tissue. There was no time-related change of this fibrosis within the glandular tissue taken between 9 and 88 months after transplantation. The content of smooth muscle actin (ASMA) was not reduced in the transplanted tissue (2.13%) compared with the control-tissue (1.13%). So the so-called ‘‘ASMA-loss’’ as indicator for degenerative processes in salivary gland tissue was not evident at all. Immunohistochemical investigation showed proliferating acinar and ductal cells in all sections (using the Ki-67index). Ki-67-antigen is not expressed in ‘‘resting cells’’ in the G-0-phase, but exclusively in proliferating cells (Fig. 13). Neuronal structures including ganglion cells
Fig. 9 e Sialoscintigraphy 5 min after injection of tracer with marked ROIs: ROI 1 ¼ transplanted gland; ROI 2 ¼ parotid gland; ROI 3 ¼ submandibular gland; ROI 4 ¼ background.
and nerve fibres were seen in all areas after staining with antibodies against PGP 9.5 as a neuron specific protein, structurally and immunologically distinct from neurone specific enolase. Protein S-100 is expressed in Schwann cells, and antibodies can show the presence of nerve fibres. Ganglion cells and nerve fibres were evident in all fields in the same amount as in the control tissue (Figs. 11 and 12).
232 Journal of Cranio-Maxillofacial Surgery
Fig. 12 e Protein S-100 reaction in transplanted glandular tissue with selective staining of Schwann cells showing nerve fibres, marked with arrows (Mayer’s haematoxylin 200).
Fig. 10 e Sialoscintigraphy 30 min after injection of Carbachol with marked ROIs: ROI 5 ¼ transplanted gland; ROI 6 ¼ parotid gland; ROI 7 ¼ submandibular gland; ROI 8 ¼ background.
Fig. 13 e Ki-67-antigen reaction with selective staining of cell nuclei of proliferating cells marked with arrows (Mayer’s haematoxylin 200).
Fig. 11 e PGP 9.5 reaction in transplanted glandular tissue with selective staining of ganglionic cells, marked with arrow (Mayer’s haematoxylin 400).
DISCUSSION Submandibular gland transfer for the correction of severe keratoconjunctivitis sicca still is rarely used to treat this common disease. This could be attributed to the complex coordination and the sophisticated surgical technique. It has been indicated only for selected severe cases in which all other established therapies had failed to stop the progressive destruction of the ocular surface.
Up to now, no investigation has been carried out to study the clinical and morphological results more than 2 years after submandibular gland transfer. Long-term evaluation was necessary to justify this treatment and its continued application. One third of the patients interviewed found no improvement in their eyes. Also objective improvement of the ocular surface occurred in less than 50% of the cases. But these data are from the first cases treated in this department by this procedure. The indication for submandibular gland transfer at that time was very rigid, so that only worst cases with severe damage of ocular surfaces were included for submandibular gland transfer. However, the slight improvement, or at least stabilisation of the ophthalmologic findings showed that it was possible to stop further progression of destruction at the ocular surface. This was considered to be worth the effort and expense. The results of the biochemical investigations showed secretions according to highly concentrated submandibular saliva in all qualities. Changes of concentration in the ‘‘saliva-tear’’ following stimulation corresponded with the changes observed in control submandibular glands. This proved the intact neurological and secretory
Long-term results of autologous submandibular gland transfer 233
mechanisms of the glands even several years after transplantation. The evidence of normal excretion of kallikrein and peroxidase especially showed the functional integrity of acinar and ductal cells. Native submandibular saliva as tear substitute has been considered as inappropriate because of its low protein content. The increased concentration of proteins and high osmolality in the secretion in this series are helpful side-effects in avoiding corneal oedema by virtue of this autologous substitute. The response to parasympathomimetics was remarkable and lead to the belief that the transplants are somehow under neuronal influence. The new sialoscintigraphic aspect highlighted in this study verified not only the evidence of vital glandular tissue in the receptor region but the evaluation of the excretory ejection fraction of labelled saliva also proved the excretory function of the transplant following stimulation: the corneal excretory function as well as the response to the muscarinic agonist, Carbachol, was demonstrated. Thus functional sialoscintigraphy seems to be an appropriate method for routine follow-up after submandibular gland transfer. Previous experimental studies reported atrophy following salivary gland denervation (Bernard, 1864; Garrett, 1987). These were experimental studies on animals; data on humans do not exist. In this series of patients, it was found that transplanted glands underwent an initial atrophy of 20% of tissue. Further degeneration or atrophy during the follow-up period of 9e 88 months was not observed. The Ki-67 index showed proliferating acinar and ductal cells in the transplants. This gives further evidence of the stable long-term results of the transplanted glands. Another interesting point was the presence of ganglionic cells and nerve fibres despite ischaemia during the transfer and year long denervation. To interpret this as a sign of re-innervation is speculative, but at least some doubt may be cast on the idea of complete independently acting tissue of those glands. It might be speculated that autonomic nerve fibres have grown along anastosed vessel walls, but this idea has to be followed up in future studies. CONCLUSION Submandibular gland transfer in most patients achieved long-term sabilisation of the ocular surface. The highly concentrated saliva improved ocular lubrication. The function proven clinically, scintigraphically and biochemically was stable for many years after surgery. The integrity of neuroception of transplanted glands provides a key point for further clinical trials to influence hypoor hyper-secretion pharmacologically.
Bohuslavizki KH, Brenner W, Lassmann S, Tinnemeyer S, Kalina S, Clausen M, Henze E: Quantitative salivary gland scintigraphy e a recommended examination prior to and after radioiodine therapy. Nuklearmedizin 36: 103e109, 1997 Collins JF, Augustin AJ: Verschiedene diagnostische Verfahren. In Collins, Augustin (Eds), Augenheilkunde. Berlin, Heidelberg: Springer, 1997: 824e825 Garrett JR: The proper role of nerves in salivary secretion: a review. J Dent Res 66: 387e397, 1987 Geerling G, Sieg P, Meyer C, Bastian G-O, Laqua H: Transplantation of autologous submandibular glands in very severe keratoconjunctivitis sicca. 2 year outcome. Ophthalmologe 95: 257e265, 1998 Hakim SG, Kosmehl H, Lauer I, Nadrowitz R, Wedel T, Sieg P: The role of myoepithelial cells in the short-term radiogenic impairment of salivary glands. An immunohistochemical, ultrastructural and scintigraphic study. Anticancer Res 22: 4121e4128, 2002 Kumar PAV, Hickey MJ, Gurusinghe CJ, O’Brien BMcC: Long term results of submandibular gland transfer for the management of xerophthalmia. Br J Plastic Surg 44: 506e508, 1991 Liem IH, Valdes Olmos RA, Balm AJM, Keus RB, van Tinteren H, Takes RP, Muller SH, Bruce AM, Hoefnagel CA, Hilgers FJ: Evidence for early and persistent impairment of salivary gland excretion after irradiation of head and neck tumours. Eur J Nucl Med 23: 397e403, 1996 Lutz J: Physiologie, anatomische Abbildungen, Embryologie und Grundbegriffe der Genetik. In Collins JF, Augustin AJ (Eds), Augenheilkunde. Berlin, Heidelberg: Springer, 1997: 839e841 MacLeod AM, Kumar PA, Hertess I, Newing R: Microvascular submandibular gland transfer: an alternative approach for total xerophthalmia. Br J Plast Surg 43: 437e439, 1990 MacLeod AM, Robbins SP: Submandibular gland transfer in the correction of dry eye. Aust N Z J Ophthalmol 20: 99e103, 1992 Moss SE, Klein R, Klein BE: Incidence of dry eye in an older population. Arch Ophthalmol 122: 369e373, 2004 Murube-del-Castillo J: Transplantation of salivary gland to the lacrimal basin. Scand J Rheumatol Suppl 61: 264e267, 1986 Schaumberg DA, Sullivan DA, Buring JE, Dana MR: Prevalence of dry eye syndrome among US women. Am J Ophthalmol 136: 318e326, 2003 Sieg P, Schirner G: Mikrovaskularer Glandula-submandibularis Transfer e eine alternative Therapie der Xerophthalmie. Dtsch Z Mund Kiefer GesichtsChir 19: 228e230, 1995 Sieg P, Geerling G, Kosmehl H, Lauer I, Warnecke K, von Domarus H: Micro vascular submandibular gland transfer for severe cases of keratoconjunctivitis sicca. Plast Reconstr Surg 106: 554e560, 2000 Wissenschaftliche Tabellen Geigy: Teilband Koerperfluessigkeiten, Traenen. 8th ed. Basel: Ciba Geigy, 1977. p. 177e180 Yu GY, Zhu ZH, Mao C, Cai ZG, Zou LH, Lu L, Zhang L, Peng X, Li N, Huang Z: Microvascular autologous submandibular gland transfer in severe cases of keratoconjunctivitis sicca. Int J Oral Maxillofac Surg 33: 235e239, 2004
Dr. Dr. Hans-Christian JACOBSEN Department of Maxillofacial Surgery University of Luebeck Ratzeburger Allee 160 23538 Luebeck Germany
References
Tel.: +49 451 500 2266 Fax: +49 451 500 4188 E-mail: [email protected]
Bernard C: Du role des actions reflexes paralysant dans le phenomene des secretions. J Anat 1: 507e513, 1864
Paper received 14 November 2005 Accepted 28 August 2007
Long-term results of autologous submandibular gland transfer for the surgical treatment of severe keratoconjunctivitis sicca Hans-Christian JACOBSEN1, Samer G. HAKIM1, Isabel LAUER2, Andreas DENDORFER3, Thilo WEDEL4, Peter SIEG1 Department of Maxillofacial Surgery (Chairman: Prof. Dr. Dr. P. Sieg), University of Luebeck, Germany; 2 Department of Radiology and Nuclear Medicine (Chairman: Prof. Dr. T. Helmberger), University of Luebeck, Germany; 3 Institute of Pharmacology and Toxicology (Chairman: Prof. Dr. P. Dominiak), University of Luebeck, Germany; 4 Institute of Anatomy (Chairman: Prof. Dr. J. Westermann), University of Luebeck, Germany
1
Aim: The aim of this study was to assess the long-term results of autologous submandibular gland transfer for surgical correction of severe keratoconjunctivitis sicca. Patients and methods: A survey was undertaken of 32 patients who had undergone submandibular gland transfer (42 glands) and by following up 11 patients (15 glands) for 5e10 years. Subjective benefit was evaluated as well as clinical findings at the ocular surface. The biochemical consistency of the secreted ‘‘saliva-tear’’ was analysed and compared with natural submandibular saliva of a matched control-group. The vitality and function of the transplants was tested by means of sialoscintigraphy. Immunohistochemical investigations were carried out in specimens of submandibular tissue, gained during reduction procedures of the transplants to correct secretory excess. Results: Patient evaluation and clinical assessment revealed a long-lasting subjective benefit in 2/3 of the patients and a stabilisation at the ocular surface in all cases. The secretion remained as highly concentrated submandibular saliva. Glandular vitality and function was shown scintgraphically. Immunohistochemical investigations revealed no progressive atrophy after transplantation, the ability of cell division remained intact and there was still neuronal tissue in all transplants, even several years after transfer. As all transplants responded well to parasympathomimetic drugs, this might be an indication of re-innervation of the gland. Ó 2008 European Association for CranioMaxillofacial Surgery
SUMMARY.
Keywords: submandibular gland transfer, keratoconjunctivitis sicca, long-term results, microvascular surgery, sialoscintigraphy
plantation of oral mucosal grafts into the conjunctival fold and/or obliteration of the lacrimal drainage pathways. A further therapeutic option was first suggested by Murube-del-Castillo in 1986, who described the microvascular autologous submandibular gland transfer for surgically correcting severe keratoconjunctivitis sicca. For complicated cases, this has been the common procedure in Luebeck for more than 10 years. This procedure requires extensive interdisciplinary coordination and is currently clinically established in only a few centres in Australia, China, the UK and Germany. Very few publications are available on the follow-up of these patients. They all describe the results within the first 2 years postoperatively (Murube-del-Castillo, 1986; Kumar et al., 1991; Sieg and Schirner, 1995; Geerling et al., 1998; Sieg et al., 2000; Yu et al., 2004). There remain several questions regarding the long-term follow-up of keratoconjunctivitis as well as the long-term stability of the function and morphology of the revascularised gland. As far as we know this paper is the first to report the long-term follow-up after submandibular gland transfer.
INTRODUCTION Dry eye syndrome is one of the most frequently occurring ophthalmological conditions. Its prevalence lies between 5 and 58% depending on selection of study groups and criteria recommended for diagnosis (Schaumberg et al., 2003; Moss et al., 2004). The development of keratoconjunctivitis sicca is proven to be associated with increasing age and female gender; alone in China about 30 million people suffer from this disorder (Yu et al., 2004). From a biological point of view, dry eye and the resulting keratoconjunctivitis is produced by the inadequate interrelation between lacrimal film and ocular surface epithelium, caused by quantitative and qualitative deficits in one or both of them. Without adequate treatment this chronic condition almost always produces permanent discomfort for the patient and is followed by a progressive destruction of the corneal surface leading to recurrent infections and visus restriction or even loss of the eye. Symptomatic treatment includes the application of tear substitutes first of all, surgical corrections such as tarsorrhaphy or im227
228 Journal of Cranio-Maxillofacial Surgery
PATIENTS AND METHODS Patients The aim was to revaluate patients who had undergone submandibular gland transfer more than 5 years ago. Out of 11 such patients, four underwent a bilateral procedure in two steps, so the total number of transferred glands was 15. The mean time between operation and evaluation was 6.4 + 0.8 years. The mean age was 53.4 + 19.4 years and eight of the 11 were female. The underlying pathology causing keratoconjunctivitis sicca was heterogeneous (Table l). Selective submandibular saliva was collected from a matched control-group of healthy patients in order to investigate the biochemical quality of the secretion and the response to pain sympathetic stimulation of the gland. Surgical technique The surgical procedure consisted of harvesting the submandibular gland with its supplying blood vessels and the excretory duct from a combined cervical and intra-oral approach (Fig. 1). The gland was transferred into a preformed pocket in the temporal muscle (Figs. 2 and 3). The blood vessels were anastomosed microsurgically to the temporal vein and artery (Fig. 4). The excretory duct was led subcutaneously to the lateral canthus and implanted into the upper lateral fornix of the conjunctiva. The key points of the procedure have already been described in detail (MacLeod et al., 1990; MacLeod and Robbins, 1992; Sieg and Schirner, 1995; Sieg et al., 2000; Yu et al., 2004). Methods Evaluation included a subjective assessment using a questionnaire, a clinical examination of the ocular surfaces and conjunctivae, a biochemical analysis of the ‘‘saliva-tears’’ in comparison with regular submandibular saliva collected from the matched control-group, and sialoscintigraphy for functional assessment of the gland. To gain more information about the structural changes in the transplants, specimens were examined from tissue taken during reduction procedures of transplanted glands. These procedures became necessary due to epiphora or persisting excessive secretion and were carried out 9e88 months (mean 26.5 months) after transplantation. Specimens from 11 cases were investigated immunohistochemically and compared with six specimens of nontransplanted submandibular tissue, removed during neck dissection procedures. Patient questionnaire and clinical evaluation The patients were sent a questionnaire including the question whether they would undergo the procedure again considering the present result. Further questions evaluated factors such as physical activity, warm temperature, and the effect of wind or chewing on the secretion as well as the changes of secretion during the daytime.
These questions had to be rated semi-quantitatively with values from 0 to 10 points. Clinical evaluation consisted of measuring the basal secretion using Schirmer’s test (Collins and Augustin, 1997). In order to evaluate the stimulation of secretion, a parasympathetic drug (0.2 mg Carbachol) was administered subcutaneously and Schirmer’s test was repeated after 10 min. The ocular surfaces were evaluated by slit-lamp examination after rose Bengal staining. These results were compared with the findings made prior to transplantation. Biochemical analysis The saliva-tears were collected before and after medical parasympathetic stimulation. They were examined for sodium, chloride, calcium phosphate and the whole protein content; and the osmolality was also evaluated. Specific proteins like kallikrein, peroxidase and secretory immunoglobulin A (slgA) were analysed to show the function of relevant cell-types in the transferred glands. Sialoscintigraphy Salivary gland scintigraphy was performed using a double head gamma camera (Picker/Marconi Prism 2000 S, Marconi Medical Systems, Cleveland, USA) with low energy ultra high resolution collimator. Directly after administering 50 MBq 99mTc-pertechnetate intravenously, dynamic scintigrams of the head (lateral views) were acquired over a period of 45e 50 min (1 frame/min, matrix 128 128, zoom factor 1.3). Salivary excretion was stimulated by subcutaneous administration of 0.2 mg Carbachol after 20 min. In order to analyse the scintigrams, regions of interest (ROIs) of the transplants were marked and time activity curves were generated (Figs. 9 and 10). Percentage tracer uptake before and after administration of Carbachol was calculated using an additional background ROI (Uptake before Carbachol Upbefore, Uptake after Carbachol Upafter). The salivary excretion fraction (EF) was quantified according to the following formula (Liem et al., 1996; Bohuslavizki et al., 1997): EFð%Þ ¼ ðUpbefore Upafter =Upbefore Þ 100. The EF of the parotid gland and, if possible, of the submandibular gland of the opposite side was calculated in the same way. Histology and immunohistochemistry The existing biopsy samples were cut at 2e6 mm and dewaxed. The amount of fat and fibrosis was measured (in %) in all sections after digital binary separation using the soft-imaging system ANALYSISÒ. Standardised immunohistochemical staining with monoclonal antibodies (DakoÔ, Glostrup, Denmark) was performed. The staining with alpha smooth muscle actin (ASMA) for the myofilaments in smooth muscle cells was an indicator for degenerative processes in salivary gland tissue (Hakim et al., 2002). Monoclonal Ki 67 antibodies (clone MIB 1) were applied to find proliferating cells in the (transferred) gland parenchyma. The reaction to protein gene product (PGP) 9.,5 and protein S-100 antibody showed the presence of neuronal ganglionic cells and nerve fibres, respectively. As chromogen a commercial
Long-term results of autologous submandibular gland transfer 229 Table l e Underlying causes of keratoconjunctivitis sicca Causes of keratoconjunctivitis sicca Patients (n ¼ 11) Traumatic loss of lacrimal gland Facial palsy nerve Benign cicatrical pemphigoid Lyell syndrome Congential aplasia of gland Idiopathic disease
1 2 1 2 1 4
Fig. 2 e Receptor area in the left temporal region. Fig. 1 e Harvested submandibular gland with supplying vessels and excretory duct.
kit with marking antibodies (ACE-KITÒ by ZymedÔ, Munich, Germany) was used. The analysis was performed at 100 magnification with a ZeissÒ graticule with 10 10 grids. RESULTS Patient questionnaire The results of the questionnaire showed that 53% of the patients were satisfied with the long-term result of the procedure, 13% were indifferent and 33% felt that the subjective outcome was not satisfactory when compared with the effort of treatment (Fig. 5). Fifty percentage of patients reported phases of hypo-secretion and 65% of hyper-secretion during the day, indicating that both inadequate conditions could occur in the same patient. During the day the secretion decreased by about 50% on average. Factors increasing secretion were warmth and physical activity (Fig. 6). Clinical evaluation The basal secretion of 4.5 ^ 4.9 mm mean as evaluated by the Schirmer’s test was increased significantly ( p ¼ 0.012) to 14.7 ^ 15.9 mm by stimulation with Carbachol (Fig. 7). There was whilst ophthalmologic examination a distinct reduction in the use of artificial tears in 30%
Fig. 3 e Prepared pocket with temporal vessels.
230 Journal of Cranio-Maxillofacial Surgery Factors influencing secretion 10 9 8 7 6 5 4 3 2 1 0 Warm temp.
Wind
Chewing
Physical activity
Fig. 6 e Factors influencing the secretion as taken from the questionnaire, rated semi-quantitatively with values from 0 to 10 points for surrounding temperature, wind, chewing and physical activity.
Fig. 4 e Transplanted gland with anastomosed vessels.
Fig. 7 e Schirmer test results (medians) from 15 transplants in mm, at rest and 10 min after stimulation with Carbachol.
of the cases revealed that the vision had not changed significantly after the surgical procedure. The cornea showed a significant improvement of lubrication in about 45% of patients.
significantly increased in the ‘‘saliva-tears’’ except for calcium and phosphate which showed a reduction. The changes of concentration on stimulation with Carbachol were similar to the changes in the secretion of the control-glands. The relatively increased concentrations of kallikrein, peroxidase and slgA as well as the changes following stimulation with Carbachol showed the specific secretory ability of acinar and ductal cells in the transplanted glands (Table 2). The essential differences between chemical composition of native submandibular saliva, the secretion of the transplanted gland and physiological tear fluid are listed in Table 3 for electrolytes, osmolality and total protein (Lutz, 1997).
Biochemical investigation
Sialoscintigraphy
Data from the biochemical analysis of the ‘‘saliva-tears’’ produced with and without stimulation were compared with the native saliva of the matched control-group. The concentrations of all investigated components were
Vitality and excretion capacity of transplanted glands could also be demonstrated by using sialoscintigraphy (Fig. 9). Excretory function was shown by stimulating the glands with Carbachol and calculating the salivary
Fig. 5 e Subjective benefit of treatment as taken from the questionnaire.
Long-term results of autologous submandibular gland transfer 231 Table 2 e Differences in concentration of salivary biochemical components between transplants and control-glands and changes following stimulation with Carbachol Biochemical concentrations
Sodium Chloride Calcium Phosphate Osmolality Total protein Kallikrein Peroxidase sIgA
Secretion of transplant compared with control
Changes in transplant secretion following stimulation with CarbacholÒ
+ + + + + + +
+ + ^
Fig. 8 e Time activity curve of tracer in the region of the 10 transplants, showing the uptake after technetium tracer injection and clearance after stimulation with Carbachol versus time.
Table 3 e Differences of chemical composition between normal tear fluid, physiological submandibular saliva and transplant secretion Chemical composition of lacrimal and salivary secretion
Sodium (mmol/1) Chloride (mmol/1) Calcium (mmol/1) Phosphate (mmol/1) Osmolality (mosmol/kg) Total protein (g/1)
Lacrimal fluid (Data from the literature)
Submandibular saliva (control group)
Transplant secretion (patients)
146* 128* 0.57* 0.07y 326* 6.69*
14 19.6 1.48 4.12 44.75 0.31
28.25 64 2.23 2.62 71.75 0.5
* Wissenschaftliche Tabellen Geigy (1977). y Lutz (1997).
production following stimulation. All transferred glands displayed a significant decrease of tracer uptake 5 min after injection of Carbachol (Fig. 8). The late scan 25 min after stimulation showed also the accumulation of tracer around the eye and in the mouth as a sign that the tracer was eliminated by secretion (Fig. 10). This proved again the response of parasympathomimetic neuroreceptors in the transplants. Histological findings Morphometry showed an average of 30% of fatty tissue and fibrosis in the transplants against 10% in the control tissue. There was no time-related change of this fibrosis within the glandular tissue taken between 9 and 88 months after transplantation. The content of smooth muscle actin (ASMA) was not reduced in the transplanted tissue (2.13%) compared with the control-tissue (1.13%). So the so-called ‘‘ASMA-loss’’ as indicator for degenerative processes in salivary gland tissue was not evident at all. Immunohistochemical investigation showed proliferating acinar and ductal cells in all sections (using the Ki-67index). Ki-67-antigen is not expressed in ‘‘resting cells’’ in the G-0-phase, but exclusively in proliferating cells (Fig. 13). Neuronal structures including ganglion cells
Fig. 9 e Sialoscintigraphy 5 min after injection of tracer with marked ROIs: ROI 1 ¼ transplanted gland; ROI 2 ¼ parotid gland; ROI 3 ¼ submandibular gland; ROI 4 ¼ background.
and nerve fibres were seen in all areas after staining with antibodies against PGP 9.5 as a neuron specific protein, structurally and immunologically distinct from neurone specific enolase. Protein S-100 is expressed in Schwann cells, and antibodies can show the presence of nerve fibres. Ganglion cells and nerve fibres were evident in all fields in the same amount as in the control tissue (Figs. 11 and 12).
232 Journal of Cranio-Maxillofacial Surgery
Fig. 12 e Protein S-100 reaction in transplanted glandular tissue with selective staining of Schwann cells showing nerve fibres, marked with arrows (Mayer’s haematoxylin 200).
Fig. 10 e Sialoscintigraphy 30 min after injection of Carbachol with marked ROIs: ROI 5 ¼ transplanted gland; ROI 6 ¼ parotid gland; ROI 7 ¼ submandibular gland; ROI 8 ¼ background.
Fig. 13 e Ki-67-antigen reaction with selective staining of cell nuclei of proliferating cells marked with arrows (Mayer’s haematoxylin 200).
Fig. 11 e PGP 9.5 reaction in transplanted glandular tissue with selective staining of ganglionic cells, marked with arrow (Mayer’s haematoxylin 400).
DISCUSSION Submandibular gland transfer for the correction of severe keratoconjunctivitis sicca still is rarely used to treat this common disease. This could be attributed to the complex coordination and the sophisticated surgical technique. It has been indicated only for selected severe cases in which all other established therapies had failed to stop the progressive destruction of the ocular surface.
Up to now, no investigation has been carried out to study the clinical and morphological results more than 2 years after submandibular gland transfer. Long-term evaluation was necessary to justify this treatment and its continued application. One third of the patients interviewed found no improvement in their eyes. Also objective improvement of the ocular surface occurred in less than 50% of the cases. But these data are from the first cases treated in this department by this procedure. The indication for submandibular gland transfer at that time was very rigid, so that only worst cases with severe damage of ocular surfaces were included for submandibular gland transfer. However, the slight improvement, or at least stabilisation of the ophthalmologic findings showed that it was possible to stop further progression of destruction at the ocular surface. This was considered to be worth the effort and expense. The results of the biochemical investigations showed secretions according to highly concentrated submandibular saliva in all qualities. Changes of concentration in the ‘‘saliva-tear’’ following stimulation corresponded with the changes observed in control submandibular glands. This proved the intact neurological and secretory
Long-term results of autologous submandibular gland transfer 233
mechanisms of the glands even several years after transplantation. The evidence of normal excretion of kallikrein and peroxidase especially showed the functional integrity of acinar and ductal cells. Native submandibular saliva as tear substitute has been considered as inappropriate because of its low protein content. The increased concentration of proteins and high osmolality in the secretion in this series are helpful side-effects in avoiding corneal oedema by virtue of this autologous substitute. The response to parasympathomimetics was remarkable and lead to the belief that the transplants are somehow under neuronal influence. The new sialoscintigraphic aspect highlighted in this study verified not only the evidence of vital glandular tissue in the receptor region but the evaluation of the excretory ejection fraction of labelled saliva also proved the excretory function of the transplant following stimulation: the corneal excretory function as well as the response to the muscarinic agonist, Carbachol, was demonstrated. Thus functional sialoscintigraphy seems to be an appropriate method for routine follow-up after submandibular gland transfer. Previous experimental studies reported atrophy following salivary gland denervation (Bernard, 1864; Garrett, 1987). These were experimental studies on animals; data on humans do not exist. In this series of patients, it was found that transplanted glands underwent an initial atrophy of 20% of tissue. Further degeneration or atrophy during the follow-up period of 9e 88 months was not observed. The Ki-67 index showed proliferating acinar and ductal cells in the transplants. This gives further evidence of the stable long-term results of the transplanted glands. Another interesting point was the presence of ganglionic cells and nerve fibres despite ischaemia during the transfer and year long denervation. To interpret this as a sign of re-innervation is speculative, but at least some doubt may be cast on the idea of complete independently acting tissue of those glands. It might be speculated that autonomic nerve fibres have grown along anastosed vessel walls, but this idea has to be followed up in future studies. CONCLUSION Submandibular gland transfer in most patients achieved long-term sabilisation of the ocular surface. The highly concentrated saliva improved ocular lubrication. The function proven clinically, scintigraphically and biochemically was stable for many years after surgery. The integrity of neuroception of transplanted glands provides a key point for further clinical trials to influence hypoor hyper-secretion pharmacologically.
Bohuslavizki KH, Brenner W, Lassmann S, Tinnemeyer S, Kalina S, Clausen M, Henze E: Quantitative salivary gland scintigraphy e a recommended examination prior to and after radioiodine therapy. Nuklearmedizin 36: 103e109, 1997 Collins JF, Augustin AJ: Verschiedene diagnostische Verfahren. In Collins, Augustin (Eds), Augenheilkunde. Berlin, Heidelberg: Springer, 1997: 824e825 Garrett JR: The proper role of nerves in salivary secretion: a review. J Dent Res 66: 387e397, 1987 Geerling G, Sieg P, Meyer C, Bastian G-O, Laqua H: Transplantation of autologous submandibular glands in very severe keratoconjunctivitis sicca. 2 year outcome. Ophthalmologe 95: 257e265, 1998 Hakim SG, Kosmehl H, Lauer I, Nadrowitz R, Wedel T, Sieg P: The role of myoepithelial cells in the short-term radiogenic impairment of salivary glands. An immunohistochemical, ultrastructural and scintigraphic study. Anticancer Res 22: 4121e4128, 2002 Kumar PAV, Hickey MJ, Gurusinghe CJ, O’Brien BMcC: Long term results of submandibular gland transfer for the management of xerophthalmia. Br J Plastic Surg 44: 506e508, 1991 Liem IH, Valdes Olmos RA, Balm AJM, Keus RB, van Tinteren H, Takes RP, Muller SH, Bruce AM, Hoefnagel CA, Hilgers FJ: Evidence for early and persistent impairment of salivary gland excretion after irradiation of head and neck tumours. Eur J Nucl Med 23: 397e403, 1996 Lutz J: Physiologie, anatomische Abbildungen, Embryologie und Grundbegriffe der Genetik. In Collins JF, Augustin AJ (Eds), Augenheilkunde. Berlin, Heidelberg: Springer, 1997: 839e841 MacLeod AM, Kumar PA, Hertess I, Newing R: Microvascular submandibular gland transfer: an alternative approach for total xerophthalmia. Br J Plast Surg 43: 437e439, 1990 MacLeod AM, Robbins SP: Submandibular gland transfer in the correction of dry eye. Aust N Z J Ophthalmol 20: 99e103, 1992 Moss SE, Klein R, Klein BE: Incidence of dry eye in an older population. Arch Ophthalmol 122: 369e373, 2004 Murube-del-Castillo J: Transplantation of salivary gland to the lacrimal basin. Scand J Rheumatol Suppl 61: 264e267, 1986 Schaumberg DA, Sullivan DA, Buring JE, Dana MR: Prevalence of dry eye syndrome among US women. Am J Ophthalmol 136: 318e326, 2003 Sieg P, Schirner G: Mikrovaskularer Glandula-submandibularis Transfer e eine alternative Therapie der Xerophthalmie. Dtsch Z Mund Kiefer GesichtsChir 19: 228e230, 1995 Sieg P, Geerling G, Kosmehl H, Lauer I, Warnecke K, von Domarus H: Micro vascular submandibular gland transfer for severe cases of keratoconjunctivitis sicca. Plast Reconstr Surg 106: 554e560, 2000 Wissenschaftliche Tabellen Geigy: Teilband Koerperfluessigkeiten, Traenen. 8th ed. Basel: Ciba Geigy, 1977. p. 177e180 Yu GY, Zhu ZH, Mao C, Cai ZG, Zou LH, Lu L, Zhang L, Peng X, Li N, Huang Z: Microvascular autologous submandibular gland transfer in severe cases of keratoconjunctivitis sicca. Int J Oral Maxillofac Surg 33: 235e239, 2004
Dr. Dr. Hans-Christian JACOBSEN Department of Maxillofacial Surgery University of Luebeck Ratzeburger Allee 160 23538 Luebeck Germany
References
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Bernard C: Du role des actions reflexes paralysant dans le phenomene des secretions. J Anat 1: 507e513, 1864
Paper received 14 November 2005 Accepted 28 August 2007