Poor secretion of transplanted submandibular glands (SMGs) during the latent period may cause duct obstruction and affects the surgical outcome. The objective of this study was to investigate the efficacy and systemic side effects of carbachol on transplanted SMG secretion. Twenty-seven patients who underwent SMG transplantation for severe keratoconjunctivitis sicca were treated with subcutaneous injections of 0.2 mg/2 ml carbachol at 10 days, 1 month, and/or 3 months after surgery. The effect on secretion was evaluated by Schirmer test and technetium 99m ( 99m Tc) scintigraphy. Systemic side effects were evaluated subjectively using a questionnaire. The results showed that the time to onset varied from 4 to 9 min and the duration of action from 50 to 110 min after carbachol administration. The secretion at each time point after drug administration was significantly higher than the pre-administration value (all P < 0.01). 99m Tc scintigraphy showed a decline in the dynamic time–activity curve in 26 patients, demonstrating a stimulatory effect on the secretion of carbachol. No serious systemic side effects were experienced. In conclusion, the intermittent administration of carbachol could be an effective and safe strategy to promote secretion from transplanted SMGs in the latent period to prevent duct obstruction.
Keratoconjunctivitis sicca (KCS) is a common disease caused by systemic or optical disorders. Severe KCS can cause corneal ulceration, opacification, or even blindness. Microvascular autologous transplantation of the submandibular gland (SMG) has proved to be an effective treatment for severe cases of KCS. Previous work by this team and others has shown that long-term secretory function is retained and the symptoms of KCS are relieved in patients who undergo SMG transplantation procedures.
Clinical observations indicate that gland secretion decreases during the first 5–7 days after transplantation, but recovers spontaneously after 3 months. These 3 months include a period of low-level secretion known as the latent period. This period is typically characterized by very low secretion and the occasional secretion of viscous fluid in the resting condition. In a pathological process similar to that of obstructive sialadenitis, duct obstruction after SMG transplantation may occur as a result of mucus emboli formed from the trapped viscous fluid. This complication can result in insufficient lubrication of the treated eyes. This team has previously reported the incidence of obstructive sialadenitis of the transplanted SMG to be 9.3% within 3 months of transplantation and 1.3% in the long term, i.e., at more than 1 year after surgery. Some patients require reoperation to relieve the obstruction, without which the transplantation is deemed a failure.
To improve surgical outcomes and the quality of life of patients with severe KCS undergoing autologous SMG transplantation, an intervention during the latent period is of critical importance.
Normal SMG secretion is controlled by cholinergic parasympathetic and sympathetic nerves. However, both of these nerve types are incised during transplantation; thus, the transplanted SMG is completely denervated. Some studies have confirmed that the parasympathetic and sympathetic nerve fibres are restored in the long term, thereby demonstrating that reinnervation occurs ; however, reinnervation-induced secretion is not found during the latent period.
Using a rabbit model of SMG transplantation, this team has previously shown that in the absence of acetylcholine, the expression levels of the muscarinic acetylcholine receptors M1 and M3 are reduced in the early stages after surgery. In addition, carbachol has been found to increase salivary secretion and prevent functional and structural injuries of the transplanted SMGs by increasing M1 and M3 receptor expression. These findings indicate that carbachol treatment could serve as a novel clinical strategy to improve secretion and prevent duct obstruction of the transplanted SMGs during the latent period.
Carbachol is a commercially available parasympathetic mimetic agent that is commonly administered via retrobulbar injection during eye surgery, or via systemic injection to treat uroschesis, postoperative abdominal distension, and xerostomia. van Acker et al. used carbachol to stimulate salivary gland secretion during single photon emission computed tomography (SPECT) to determine the relationship between radiation dose and salivary gland dysfunction after radiotherapy. Geerling et al. also suggested the use of carbachol to stimulate gland secretion during salivary gland scintigraphy for transplanted SMGs. However, no studies have reported the use of carbachol to promote SMG secretion and to prevent obstructive sialadenitis of the transplanted SMGs.
The objectives of this study were to investigate whether carbachol could promote the secretion of transplanted SMGs during the latent period via systemic administration and to identify its systemic side effects, if any.
Materials and methods
Twenty-seven patients (11 men and 16 women) with a mean age of 37.5 years (range 17–51 years) who underwent autologous SMG transplantation between May 2008 and December 2012 were enrolled in this study. The aetiologies of KCS were Stevens–Johnson syndrome in 18 patients, acute conjunctivitis in three patients, corneal pemphigoid in one patient, corneal chemical burns in one patient, and unknown in four patients. KCS status was evaluated by an ophthalmologist using the Schirmer test, break-up time, and fluorescence staining. The indications for SMG transplantation included the following: (1) obvious, persistent symptoms of dry eye and failure of other ophthalmological treatments, and (2) Schirmer test result <2 mm/5 min, break-up time result <5 s, and fluorescence staining (score value ≥10) on ophthalmological evaluation. Contradictions to SMG transplantation included (1) obvious symptoms of xerostomia or Sjögren syndrome, (2) Schirmer test result >5 mm/5 min, and (3) hypofunction of multiple major salivary glands on scintigraphy.
No control group was set in this study for two reasons. Firstly, in the authors’ long term clinical experience, secretion in the latent period is very low and the patient’s general physical condition rarely affects the secretion from the transplanted SMG. Secondly, results from animal studies and a pilot study on patients found that carbachol was very effective on the secretion of the transplanted SMG and therefore very unlikely to be affected by other factors, if any exist. The study was approved by the necessary institutional review board. All of the patients signed an informed consent form before treatment.
Administration of carbachol
All patients were injected subcutaneously with 0.2 mg/2 ml carbachol (Shandong Bausch and Lomb Freda Pharmaceutical Co., Ltd, Jinan, China) bilaterally in the abdomen, 5 cm below the navel, on day 10 postoperative and at 1 month and/or 3 months after autologous SMG transplantation. All patients were subject to electrocardiographic monitoring during carbachol administration. Atropine was available for administration in the event of severe cardiovascular symptoms or other severe systemic side effects.
No carbachol injection was given if the patient’s resting Schirmer test result was >10 mm/5 min or if epiphora was reported; this was set as the endpoint of the treatment.
Schirmer test procedure
A Schirmer test was performed before (baseline) and at 5, 10, 15, 30, and 60 min after drug administration. The room temperature was maintained at 23 °C and patients were asked to refrain from any physical activity or glandular stimulation for 30 min to prevent these affecting the flow rate. When the secretion rate was moderate, it was measured according to a standard Schirmer test protocol with no anaesthesia. The bent end of a Whatman No. 41 paper strip (5 mm × 120 mm) was inserted into the lateral side of the lower conjunctival fornix for 5 min and the length of the wet strip was recorded. When the secretion rate was high, the fluid secreted in 5 min was collected in Eppendorf tubes. The collected salivary tears were then dropped onto a Whatman paper strip using a pipette slowly and evenly. The length of the wet strip was recorded. The values were recorded as ST1 (baseline) and ST2–ST6 for the subsequent time points. A stop watch was used to record the onset time and end point. Onset time was defined as the time when visible salivary secretion could be seen from the conjunctival sac. Additionally, beyond 60 min, the secretion from the transplanted SMG was observed. When it appeared to subside, it was tested continuously until it was less than or equal to that at baseline. The time taken to achieve this was recorded as the duration of action.
Evaluation by technetium 99m ( 99m Tc) scintigraphy
In addition to the Schirmer test, 99m Tc scintigraphy was used to evaluate the efficacy of carbachol before surgery and at 7 days and at 3 months after surgery. 99m Tc scintigraphy was performed using Hawkeye SPECT equipment (GE Healthcare, Tirat Hacarmel, Israel). Patients fasted for at least 8 h before the examination. During the whole procedure, patients were positioned using a radiolucent and neck-contoured headrest made of plastic. After intravenous injection of the tracer 99m Tc pertechnetate, sequential images were acquired for 30 min at a rate of 1 min/frame using a gamma camera. At 15 min, 0.2 mg/2 ml carbachol was administered subcutaneously. Throughout the study, the camera was fitted with a low-energy ultra high resolution (LEUHR) collimator. Regions of interest (ROIs) were drawn by overlaying all frames of transplanted SMGs and the contralateral SMGs, and the background ROI was marked in the frontal region. Dynamic time–activity curves were generated automatically by the workstation after all ROIs were drawn ( Fig. 1 ).
Three parameters were used to investigate the secretion efficiency of transplanted SMGs in response to carbachol, namely the uptake ratio (UR), salivary excretion fraction (SEF), and latent time. UR was defined as the ratio between maximum radiation counts and the background radiation counts. The UR is a measure of the uptake function of acinar cells. The SEF is defined as the ratio of the difference between the maximum and minimum radiation counts to the maximum radiation counts. It is a measure of the stimulatory effect of carbachol on acinar cells. The latent time is defined as the time interval between carbachol administration and excretion, and it represents the speed at which acinar cells respond to carbachol. As latent time is affected by various parameters, such as the thickness of the subcutaneous fat tissue, heart rate, injection rate, etc., the latency index was also obtained. The latency index is the ratio between the onset time of transplanted SMGs and the average onset time of bilateral parotid glands.
Radiation counts for maximum and minimum activity after carbachol injections were denoted as ‘a’ and ‘b’, radiation counts in the background area were denoted as ‘c’. The time point of the maximum radiation count was Ta. Accordingly, the UR, SEF, latent time, and latent index of the transplanted SMGs were calculated as follows:
Latent index = 2 × T latent transplanted SMG / ( T latent left parotid gland + T latent right parotid gland )