the recurrent laryngeal nerve ( rln ) injury is one of the most important complications during thyroid surgery , leading to dysfunction , and palsy of the vocal cords . for years , surgeons have attempted to prevent this complication by improving surgical techniques and optimizing surgical strategies .
the identification and preservation of the anatomical integrity of the rln during thyroid surgery is commonly accepted as a  gold standard .
 however , even in the most experienced hands , rln injury occasionally occurs . in the last decade , intraoperative neuromonitoring ( ionm ) has been widely applied during thyroid surgery to aid in the localization and identification of the rln .
one of the most important roles of ionm is assistance in intraoperative navigation and rapid identification of rln distribution .
repetitive tissue stimulation in the vicinity of the nerve allows the nerve to be localized using a stimulation probe before visual identification . the reported stimulation intensity for mapping the path of the nerve ranges from 2 to 3 ma . this range is higher than that used for direct nerve stimulation , as the use of stronger stimulation current would depolarize a greater sphere of tissue around the probe tip
however , failure to obtain signal deflection at 3 ma during nerve localization might occasionally occur , in which case a surgeon might consider increasing the stimulation intensity . however , electrical stimulation is also a potential risk factor for rln injury , and the nerve can become damaged in the case of over - stimulation .
therefore , in this study , we developed an experimental canine model to determine whether supramaximal stimulation during ionm could induce nerve damage and investigated the safe range of stimulation current intensity .
twenty adult , male beagle dogs weighing 1520 kg were obtained from the animal center of peking university health science center ( institutional protocol no .
the dogs were randomly divided into four groups of five each and maintained under standard laboratory conditions with free access to food and water .
all animal experiments were approved by the institutional ethics committee ( peking university people 's hospital ) , and all procedures were conducted according to our institutional animal care guidelines . the animals were fasted for 8 h and then anesthetized with intravenous 3% pentobarbital ( merck kgaa , darmstadt , germany ) 5 ml and vecuronium ( organon pharmaceutical co. , ltd .
, oss , the netherlands ) 0.06 mg / kg , after which a neural monitoring tube ( medtronic nim 2 emg ett # 6 ; medtronic , jacksonville , florida , usa ) was inserted by a single anesthesiologist .
, new york , usa ) to ensure that the middle of its blue - marked region ( the exposed electrodes ) formed a good contact with the true vocal cords .
the tube was then fixed by balloon inflation . after performing routine surgical procedures such as disinfection and draping , a midline vertical cervical incision was made to expose the neck and the larynx , and total thyroidectomy was performed .
a nerve integrity monitor ( nim - response 2.0 ; medtronic , jacksonville , florida , usa ) was applied for ionm approximately 20 min after the administration of muscle relaxant , at the moment of muscle recovery .
the following parameters were set : stimulation rate , four times / s ; pulse width , 100 s ; and event threshold , 100 s . while exposing the bilateral rln , a stimulation current was delivered to the nerves by a bipolar probe at a 30 hz frequency .
the electrode detected evoked electromyography ( emg ) activity , the parameters of which were recorded . after finishing the experimental procedures ,
the incisions were closed in multiple layers , and the animals were sacrificed by an intravenous bolus injection of potassium chloride .
all surgeries were performed by the same surgeon , who is experienced in thyroid surgery .
the stimulation threshold was defined as the lowest current required to obtain clear evoked emg .
each nerve was initially stimulated using a 0.1 ma current , and the current was increased step - wise by 0.05 ma until emg was observed .
the current was recorded as the stimulation threshold , and the corresponding amplitude was recorded as well .
after exposure of the rln , the stimulation threshold and amplitude at the threshold level were measured as baseline parameters before any interventions .
then , the rlns were continuously stimulated with a high - intensity current for 1 min at a point distal from the test point .
each group of animals was exposed to a different stimulation intensity ( 5 ma , 10 ma , 15 ma , and 20 ma ) .
the stimulation threshold and evoked emg amplitude were measured 1 min later at the same point as the prior test to determine poststimulation parameters .
all interventions and recordings were performed completely on one side before treating the other side .
the specimens were prepared using the following steps : ( 1 ) immediate fixing in 3% glutaraldehyde ( sigma - aldrich co. llc . , st .
louis , usa ) ; ( 2 ) three washes of 20 min each with phosphate - buffered solution ( zoman bio co. , ltd . , beijing , china ) ;
( 3 ) fixing in osmic acid ( alfa aesar , massachusetts , usa ) for 1.52.0 h ; ( 4 ) two washes with distilled water ( sinopharm chemical reagent beijing co. , ltd , beijing , china ) ; ( 5 ) immersion in uranium ( merck kgaa , darmstadt , germany ) for 30 min ; ( 6 ) two washes with distilled water ; ( 7 ) dehydration with graded ethanol ( beijing chemical works , beijing , china ) ( 50% , 70% , 90% , and 100% for 15 min each ) ; ( 8) embedding in 1:1 100% acetone ( beijing chemical works , beijing , china ) under vacuum for 23 h ; ( 9 ) embedding in 1:3 100% acetone : under vacuum for 23 h ; ( 10 ) embedding and immersion at 37c for 1620 h ; ( 11 ) polymerization at 60c for 48 h ; and ( 12 ) drying for 24 h. finally , the myelin sheath , neuraxon , and schwann cell nerve microstructures were observed under an electron microscope ( jeol ltd . ,
data were analyzed using spss 17.0 statistical software ( spss inc . , chicago , il , usa ) .
paired t - tests were used to compare the average stimulation thresholds and amplitudes at the threshold level between normal and stimulated rlns .
twenty adult , male beagle dogs weighing 1520 kg were obtained from the animal center of peking university health science center ( institutional protocol no .
the dogs were randomly divided into four groups of five each and maintained under standard laboratory conditions with free access to food and water .
all animal experiments were approved by the institutional ethics committee ( peking university people 's hospital ) , and all procedures were conducted according to our institutional animal care guidelines .
the animals were fasted for 8 h and then anesthetized with intravenous 3% pentobarbital ( merck kgaa , darmstadt , germany ) 5 ml and vecuronium ( organon pharmaceutical co. , ltd .
, oss , the netherlands ) 0.06 mg / kg , after which a neural monitoring tube ( medtronic nim 2 emg ett # 6 ; medtronic , jacksonville , florida , usa ) was inserted by a single anesthesiologist .
, new york , usa ) to ensure that the middle of its blue - marked region ( the exposed electrodes ) formed a good contact with the true vocal cords .
after performing routine surgical procedures such as disinfection and draping , a midline vertical cervical incision was made to expose the neck and the larynx , and total thyroidectomy was performed .
a nerve integrity monitor ( nim - response 2.0 ; medtronic , jacksonville , florida , usa ) was applied for ionm approximately 20 min after the administration of muscle relaxant , at the moment of muscle recovery .
the following parameters were set : stimulation rate , four times / s ; pulse width , 100 s ; and event threshold , 100 s . while exposing the bilateral rln , a stimulation current was delivered to the nerves by a bipolar probe at a 30 hz frequency .
the electrode detected evoked electromyography ( emg ) activity , the parameters of which were recorded . after finishing the experimental procedures ,
the incisions were closed in multiple layers , and the animals were sacrificed by an intravenous bolus injection of potassium chloride .
all surgeries were performed by the same surgeon , who is experienced in thyroid surgery .
the stimulation threshold was defined as the lowest current required to obtain clear evoked emg .
each nerve was initially stimulated using a 0.1 ma current , and the current was increased step - wise by 0.05 ma until emg was observed .
the current was recorded as the stimulation threshold , and the corresponding amplitude was recorded as well .
after exposure of the rln , the stimulation threshold and amplitude at the threshold level were measured as baseline parameters before any interventions .
then , the rlns were continuously stimulated with a high - intensity current for 1 min at a point distal from the test point .
each group of animals was exposed to a different stimulation intensity ( 5 ma , 10 ma , 15 ma , and 20 ma ) .
the stimulation threshold and evoked emg amplitude were measured 1 min later at the same point as the prior test to determine poststimulation parameters .
all interventions and recordings were performed completely on one side before treating the other side .
the specimens were prepared using the following steps : ( 1 ) immediate fixing in 3% glutaraldehyde ( sigma - aldrich co. llc . , st .
louis , usa ) ; ( 2 ) three washes of 20 min each with phosphate - buffered solution ( zoman bio co. , ltd . , beijing , china ) ;
( 3 ) fixing in osmic acid ( alfa aesar , massachusetts , usa ) for 1.52.0 h ; ( 4 ) two washes with distilled water ( sinopharm chemical reagent beijing co. , ltd , beijing , china ) ; ( 5 ) immersion in uranium ( merck kgaa , darmstadt , germany ) for 30 min ; ( 6 ) two washes with distilled water ; ( 7 ) dehydration with graded ethanol ( beijing chemical works , beijing , china ) ( 50% , 70% , 90% , and 100% for 15 min each ) ; ( 8) embedding in 1:1 100% acetone ( beijing chemical works , beijing , china ) under vacuum for 23 h ; ( 9 ) embedding in 1:3 100% acetone : under vacuum for 23 h ; ( 10 ) embedding and immersion at 37c for 1620 h ; ( 11 ) polymerization at 60c for 48 h ; and ( 12 ) drying for 24 h. finally , the myelin sheath , neuraxon , and schwann cell nerve microstructures were observed under an electron microscope ( jeol ltd . , tokyo , japan ) .
data were analyzed using spss 17.0 statistical software ( spss inc . , chicago , il , usa ) .
paired t - tests were used to compare the average stimulation thresholds and amplitudes at the threshold level between normal and stimulated rlns . a value of p < 0.05 was considered statistically significant .
the bilateral rlns of all 20 dogs were successfully exposed ; therefore , 10 nerves per group were available for experimentation .
the average stimulation thresholds of the unstimulated rlns in the 15 ma group and 20 ma group were 0.315  0.097 ma and 0.300  0.103 ma , and the average amplitudes at the threshold level were 1,021  273 v and 1,200  258 v , respectively .
the data from the groups stimulated with 5 ma and 10 ma were accidentally lost . after the experimental invention , no significant difference in average stimulation threshold was observed in any group [ 15 ma group : unstimulated 0.315  0.097 ma vs. stimulated 0.320  0.123 ma , p = 0.847 ; 20 ma group : unstimulated 0.300  0.103 ma vs. stimulated 0.305  0.101 ma , p = 0.758 ; table 1 ] .
similarly , there was no statistically significant change in average evoked emg amplitude in any group , even the group stimulated with 20 ma [ 15 ma group : unstimulated 1,021  273 v vs. stimulated 1,026  268 v , p = 0.834 ; 20 ma group : unstimulated 1,200  258 v vs. stimulated 1,162  275 v , p = 0.148 ; table 2 ] .
stimulation threshold changes for recurrent laryngeal nerves after electrical stimulation at different intensities sd : standard deviation .
changes in evoked electromyography amplitude of the recurrent laryngeal nerve after electrical stimulation at different intensities sd : standard deviation .
no significant pathological changes were observed among the specimens from the 5 ma , 10 ma , and 15 ma groups compared to the normal group under electron microscopy [ figure 1 ] .
aligned microfilaments , evenly distributed matrix , and normal organelles were clearly observed without schwann cell degeneration or proliferation [ figure 2 ] .
electron microscopic image of a normal recurrent laryngeal nerve ( original magnification , 10,000 ) .
electron microscopic image of a recurrent laryngeal nerve continuously stimulated with a 15 ma current for 1 min ( original magnification 10,000 ) .
electron microscopic image of a recurrent laryngeal nerve stimulated with a 20 ma current for 1 min .
the bilateral rlns of all 20 dogs were successfully exposed ; therefore , 10 nerves per group were available for experimentation .
the average stimulation thresholds of the unstimulated rlns in the 15 ma group and 20 ma group were 0.315  0.097 ma and 0.300  0.103 ma , and the average amplitudes at the threshold level were 1,021  273 v and 1,200  258 v , respectively .
the data from the groups stimulated with 5 ma and 10 ma were accidentally lost . after the experimental invention , no significant difference in average stimulation threshold was observed in any group [ 15 ma group : unstimulated 0.315  0.097 ma vs. stimulated 0.320  0.123 ma , p = 0.847 ; 20 ma group : unstimulated 0.300  0.103 ma vs. stimulated 0.305  0.101 ma , p = 0.758 ; table 1 ] .
similarly , there was no statistically significant change in average evoked emg amplitude in any group , even the group stimulated with 20 ma [ 15 ma group : unstimulated 1,021  273 v vs. stimulated 1,026  268 v , p = 0.834 ; 20 ma group : unstimulated 1,200  258 v vs. stimulated 1,162  275 v , p = 0.148 ; table 2 ] .
stimulation threshold changes for recurrent laryngeal nerves after electrical stimulation at different intensities sd : standard deviation .
changes in evoked electromyography amplitude of the recurrent laryngeal nerve after electrical stimulation at different intensities sd : standard deviation .
no significant pathological changes were observed among the specimens from the 5 ma , 10 ma , and 15 ma groups compared to the normal group under electron microscopy [ figure 1 ] .
aligned microfilaments , evenly distributed matrix , and normal organelles were clearly observed without schwann cell degeneration or proliferation [ figure 2 ] .
electron microscopic image of a normal recurrent laryngeal nerve ( original magnification , 10,000 ) .
electron microscopic image of a recurrent laryngeal nerve continuously stimulated with a 15 ma current for 1 min ( original magnification 10,000 ) .
electron microscopic image of a recurrent laryngeal nerve stimulated with a 20 ma current for 1 min .
rln injury is a major complication of thyroid surgery . such injury results in temporary or permanent recurrent laryngeal nerve palsy ( rlnp ) , which manifests as hoarseness in unilateral injury or life - threatening acute airway obstruction in bilateral injury .
temporary rlnp occurs in 1.438.4% of cases while permanent palsy occurs in 018.6% of cases .
the strategy used for thyroid surgery has advanced to routine capsular dissection and direct visualization of the rln , which is accepted as the gold standard due to its association with a significantly lower rlnp rate .
ionm was recently introduced to aid in the localization and identification of the rln and as a prognostic measure of postoperative nerve function .
early and definite identification of the rln during thyroid surgery , especially in difficult cases , is important for preventing rln injury .
previously reported stimulus intensities used to localize the nerve before visualization has ranged from 1 to 3 ma .
many studies have confirmed that the stimulation current that is routinely used is safe and meets the demands required for surgery most of the time .
however , it has been clearly demonstrated that over - stimulation induces tissue damage , which may result from the passage of current through the tissue .
the range of stimulation current intensity that can be safely applied during clinical practice remains to be determined . in this study , we used twenty dogs to evaluate the functional and histological outcomes of applying supramaximal electrical stimulation to the rln .
the safety of stimulation with 3 ma has been widely confirmed in clinical and animal experiments . in a study reported by ulmer et al .
, the maximal current used for stimulation was 5 ma , and no postoperative rlnp was detected .
however , stimulation with 5 ma during ionm has rarely been used in the clinic . in the current study , we initially treated animals with a current of 5 ma and observed no functional and histological changes after the intervention , verifying its safety .
previous studies in canine or porcine models that have characterized emg changes , such as amplitude decrements and increases in stimulation threshold level after neural injury , have suggested that these parameters can be used as predictors of nerve injury .
even after stimulation with 20 ma , there was still no functional abnormality in the nerve based on the stimulation thresholds and evoked emg amplitudes observed .
normal morphology was observed after stimulation with 15 ma , but acute microstructural morphological changes occurred after stimulation with 20 ma .
the anatomical and functional correlations of our canine model might be translatable into a safe range of stimulation intensity for ionm during human thyroid surgery . despite losing the data from the groups stimulated with 5 ma and 10 ma , based on our results
, we postulate that a stimulation intensity less than 15 ma will not induce any functional or histological changes to rlns during ionm .
we chose to evaluate a canine model because dog thyroid glands have similar anatomy to human thyroid glands and therefore can be used to mimic human thyroid surgery .
the canine thyroid is a dark red , elongated structure that is attached to the fascia along the ventrolateral surfaces of the proximal trachea , ventrally covered by the sternocephalicus and sternohyoideus muscles , and laterally covered by the sternothyroideus muscle .
the caudal laryngeal nerves in dogs , termed rlns in humans , are dorsal to the thyroid lobes .
one limitation of the current study is the loss of the data from the groups stimulated with the low - intensity current .
another limitation was the lack of exclusion of influencing factors and the absence of functional evaluation of the nerves , such as the observation of vocal cord movement by video laryngoscopy .
however , this study provides evidence for the safety of using electrical stimulation during ionm in clinical practice . in conclusion
, our study indicates that a stimulation intensity less than 15 ma could be safely applied during ionm .
 background : intraoperative neuromonitoring ( ionm ) of the recurrent laryngeal nerve ( rln ) has been widely applied during thyroid surgery . 
 however , the safe range of stimulation intensity for ionm remains undetermined.methods:total thyroidectomies were performed on twenty dogs , and their rlns were stimulated with a current of 520 ma ( step - wise in 5 ma increments ) for 1 min . 
 the evoked electromyography ( emg ) of vocal muscles before and after supramaximal stimulation were recorded and compared . 
 acute microstructural morphological changes in the rlns were observed immediately postoperatively under an electron microscope.results:the average stimulating threshold for rlns stimulated with 15 ma and 20 ma showed no significant changes compared to the unstimulated rlns ( 15 ma group : 0.320  0.123 ma vs. 0.315  0.097 ma , p = 0.847 ; 20 ma group : 0.305  0.101 ma vs. 0.300  0.103 ma , p = 0.758 ) . 
 similar outcomes were shown in average evoked emg amplitude ( 15 ma group : 1,026  268 v vs. 1,021  273 v , p = 0.834 ; 20 ma group : 1,162  275 v vs. 1,200  258 v , p = 0.148 ) . 
 however , obvious acute microstructural morphological changes were observed in the nerves that were stimulated with 20 ma.conclusions:a stimulation intensity less than 15 ma might be safe for ionm of the rln . 
I
M
Experimental animals
Preoperative preparation
Surgical procedures
Experimental design
Histological evaluation
Statistical analysis
R
Neuromonitoring outcomes
Histological outcomes
D
Financial support and sponsorship
Conflicts of interest