Experimental Study On Neural Regeneration And Effect Of Electrostimulation On It After Oculomotor Nerve Injury

PartⅠESTABLISHMENT OF THE ANIMAL MODEL WITH OCULOMOTOR NERVE INJURY AND ELECTROSTIMULATION IN BEAGLE DOGBackgroundsCraniocerebral trauma and surgical treatment of the lesions of skull base and orbital cavity frequently resulted in oculomotor nerve(OMN) injury,which severely impaired the patient's quality of life.Up to now,the rule of OMN regeneration was obscure,and the study on the effective intervention that targets neural regeneration was insufficient.As a consequence,the research on the mechanism of regeneration and functional recovery of traumatic OMN could offer theoretical and technical supports to successfully functional recovery of oculomotor and other cranial nerve. Some authors have studied the regenerative mechanism of OMN in many kinds of animal models and proved its anatomic neural regeneration and functional restoration. However,it was insufficient for the description of the surgical anatomy and approaches of intracranial OMN and exraocular muscles,and of establishment of the animal model.Moreover,the present methods used in neuroelectrostimulation and electromyographic recording in experimental and clinical study of cranial nerve regeneration were not suitable to the long-term and dynamic electrophysiological research.From an ethical,economical and available point of view,the small animal was usually used in the study on cranial nerve regeneration,but it was very difficult to perform more complicated intracranial and ophthalmic operation in them due to the limited operating field.Therefore,the large animal was naturally considered in this situation.As the basic work of our research on oculomotor nerve regeneration and on effect of electrostimulation on it,we constructed a safe and available dog model with OMN injury and elecrostimulation,and researched the fuctional and neurohistologic characteristics of OMN and the electrophysiologic features of inferior obliquus(IO) in normal dog.Objectives(1) To establish the animal model with oculomotor nerve(OMN) injury and electrostimulation.(2) To research the fuctional and neurohistologic characteristics of oculomotor nerve,and the electrophysiologic features of inferior obliquus in normal dog.Methods and materials1.Establishment of animal modelForty two beagle dogs were obtained from Shanghai Jiaotong University Agronomic College.Under General anaesthesia and sterile condition,all animals underwent a right transfrontotemperal approach to OMN,a transconjunctival route to inferior obliquus(IO) and electrode implantation.The oculomotor nerve between its exit from the midbrain and the entrance into the cavernous sinus was crushed with a 30-second complete occlusion of a gun-shaped forceps under an operating microscope. The procedure severed all axons but left the nerve sheath intact as a conduit for regeneration.Twelve dogs were subjected to only OMN injury,fifteen ones OMN injury and electrode implantation of IO,and other fifteen dogs OMN injury plus electrode implantation of OMN and IO.2.Assessment of animal modelClinical examination was performed for the animals' general state of health, mental state,operative incision and neurological function.The function of pupil sphincter muscle and of extraocular muscles was observed to assess the effects of OMN injury.The completeness of nerve crush injury was assessed by histological procedures described in the previous studies.The integrality,stability and security of the electrodes implanted on OMN and in IO were inspected everyday postoperatively, and their availability and reliability were assessed by electrophysiological method.3.Neurohistologic examinationThe whole OMN stem was removed after in situ fixation.1) The specimen was dehydrated,imbedded in paraffin,and sectioned transversely at 5μm,and then the sections were processed with HE or myelin staining,and analyzed under light microscope;or 2) Following the specimen was post-fixed,dehydrated,and implanted in Epon812,semi-thin and ultra-thin transversal sections were cut on an LKB ultramicrotome.Semi-thin sections were stained with Toluidine blue and viewed using light microscopy,whereas ultra-thin sections were double-stained with uranyl acetate and lead citrate,and viewed and photographed in a Philips CM10 transmission electron microscope.Image analysis was performed with Axioplan-2 Imaging System.4.OMN electrostimulation and electrophysiologic examination of IOThe Powerlab System(ADInstruments Pty Ltd.,Australia) was used in OMN stimulation and electromyographic examination of IO.The proximal monopolar electrode on OMN was connected to the anode of the stimulator,and the distal one to the cathode.The ground electrode was connected to the oral lip of animal with an alligator clip.The stimulation pulse was 5 Hz,0.1-ms duration and 0.5-1.8 volt,and the band pass for electromyographic record was set between 20 Hz and 10 kHz. Compound muscle action potentials(CMAPs) were memorized synchronously during OMN stimulation.Spontaneous electric activity and motor unit potentials(MUPs) were recorded while the phon-/photoinduced ocular movement occurred in the conscious animals.All the electromyographic data were stored and analyzed off-line by the software package shipped with Powerlab System.5.Data analysisData were displayed as the mean±standard deviation.Independent-samples t test was used in comparisons of means between two groups.Differences were considered significant when P＜0.05.Results1.Animals' general state of health With the above two surgical approaches,the dog intracranial OMN and IO were sufficiently revealed and implanted stimulating and recording electrode respectively, and many kinds of nerve intervention could be performed on OMN.Postoperatively, all dogs did well and had good operative incisions healing with no signs of cranial and ocular infection,corneal ulcer or neurological dysfunction except for complete right OMN paralysis due to the experimental nerve injury.2.Assessment of animal modelFor intact dogs,the pupil diameter was 3.92±0.80 mm,and light reaction was sensitive;the distance between pupil center and outer canthus(Dpc) and palpebral fissure size(PFS) was 9.97±1.11 mm and 10.91±1.37 mm respectively.All OMN-injured dogs had immediate and complete right oculomotor paralysis postoperatively.Neurohistologic examination showed all axons were severed but the nerve sheaths intact.During experiments,all implanted electrodes worked stably and effectively with no twist,brisement and exodus,and did not induce intracranial and/or ocular infection and additional OMN injury.3.Neurohistologic characteristics of dog OMNFor adult dog,the length of OMN was 1.96±0.15 cm between its exit from the midbrain and the entrance into the extraocular muscles.It consisted of myelinated and unmyelinated nerve fibers;the mean value of the total of nerve fibers was 10,282.00±1,195.66.For myelinated nerve fiber,the mean value of the number of nerve fibers was 8,875.00±1,088.72,being the 86.32±0.55%of the total of nerve fibers;the average diameter of fibers and axons was 10.61±0.72μm and 6.46±0.41μm respectively;the mean value of myelin thickness was 2.08±0.27μm.For unmyelinated nerve fiber,the mean value of the number of nerve fibers was 1,407.00±108.22,being the 13.68±0.55%of the total of nerve fibers;the average diameter of fibers was 0.45±0.23μm.4.Electrophysiologic characteristics of dog IOIn the inferior obliquus(IO) of the healthy and conscious dog,no spontaneous electric activity was recorded when the eyes in rest position.The normal MUPs had uniform waveform demonstrating 2-4 phases,duration 3.16±0.15 ms and amplitude 524.18±133.89μvolt with normal recruitment and no multiphasic MUP when the phon-/photo induced ocular movement occurred.With the supramaximal stimulation on OMN,the latency,peak-to-peak amplitude and duration of CMAPs recorded in IO was 2.91±0.03 ms,526.67±15.89μvolt and 4.28±0.21 ms respectively.Conclusions(1) The animal model of oculomotor nerve injury and electrostimulation was successfully established after the dog intracranial OMN was injured and implanted stimulating electrodes through transfrontotemperal approach,and its inferior obliquus was implanted recording electrodes via a transconjunctival route.With the method to establish this animal model,dog survival rate was high,and all animals were free from severe complications.The model was stable,reliable,and suitable to the dynamic study on many aspects of the regeneration and electrophysiology of posttraumatic OMN.(2) The basic functional and neurohistologic characteristics of oculomotor nerve and the foundational electrophysiologic features of inferior obliquus were determined in normal dog.