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Background incontinence is persistent or recurrent episodes of intestinal contents can not be perceived trait or unable to control the discharge of intestinal contents, including liquid, gaseous, solid intestinal contents, are complex, multi-factor induced bowel dysfunction, manifested for the ability to control gas or stool damage, resulting in increased frequency of defecation, often diarrhea. These symptoms often lead to patients refused to participate in social activities and seriously affect the patient's daily life. Although researchers have proposed a variety of surgical and non-surgical treatment, but each has its limitations and shortcomings, is still intractable incontinence, surgical clinic very intractable problem. To this end, we tried to develop a method of making an animal model of incontinence, fecal incontinence with expectations for the production of animal model provides a new way for the treatment of anal incontinence disease research to do basic work. In view of scholars currently used modeling methods developed new creation model approach to achieve desired main are: ① simple operation, without expensive laboratory equipment and laboratory reagents, easy to spread; ② minimally invasive operation, the animal mortality rate is low; ③ modeling the exact effect and stable. According to previous research, the external anal sphincter during defecation control plays an important role, and its innervation of the sacral nerve. Cosovic E and other studies have found high concentrations of local injection of local anesthetic drugs, can occur after nerve degeneration in one week. Local anesthetic nerve damage in a dose dependent manner, the higher the concentration, the extent of damage more severe until the motor and sensory function permanently lost. In addition, when the drug concentration reaches a critical level, the local anesthetic has detergent properties, can cause structural damage to nerve cell membranes, this process was irreversible change. Nerve local injection of ethanol volume fraction 0.99, can lead to peripheral nerve block, used for treatment of trigeminal neuralgia. Based on theoretical appeal, we initially explored a new method of making an animal model incontinence, functional positioning through the external anal sphincter innervation, local injection of neurotoxic drugs, selective destruction of the external anal sphincter innervation to effect muscle, ie the external anal sphincter dysfunction occurs, successfully fabricated incontinence in animal models. Objective Based on simple, economical and practical, minimally invasive operation, the exact effect of a stable modeling concept, incontinence developed an animal model produced new ways to expect an animal model for the production of incontinence provide a new way of thinking for the treatment of incontinence disease research to do basic work. Method 1, the experimental animals and grouping eight-month-old healthy adult male New Zealand rabbits 30, weighing 2.0-2.5 kg, raising the general level. 30 New Zealand white rabbits were randomly divided into five groups randomly, control group A (n = 6), local injection of saline, after 8 weeks after local tissue for pathological examination; experimental group B (n = 6) , local injection of 50g ╱ L ropivacaine, 4 weeks after taking the local tissue for pathological examination; experimental group C (n = 6), local injection of 50g ╱ L ropivacaine, take 8 weeks local tissue for pathological examination; experimental group D (n = 6), local injection of ethanol volume fraction of 0.99, after 4 weeks after local tissue for pathological examination; experimental group E (n = 6), local injection of the volume fraction 0.99 ethanol, after 8 weeks after local tissue for pathological examination. 2, anesthetized animals anesthetized animals in this experiment are used 30g ╱ L 1ml/kg intravenous pentobarbital anesthesia. 3, test animals under anesthesia preoperative resting anal canal pressure (anal canal resting pressure, ARP) fixed animals, and sacral hair removal around the anus, anal manometry bladders built into the connection tee, pressure calibration table pressure signal converter, sixteen physiological signal acquisition system PowerLab 16/30 (ML880), Dual Bio Amp / Stimulator (ML408), measured under anesthesia animal ARP. 4, functional positioning bilateral 4th nerve and external anal sphincter recommended line EMG (electromyography, EMG) examination from the puncture site in the more distant one indifferent electrode placement, and the puncture needle and the pulse is connected to a loop, In the external anal sphincter SRE two sensing electrodes. Will stimulate the current open to 1.5mA, pulse frequency 1 Hz. Palpable in the rabbit sacral sacral lower, in accordance with section 4 recommended locate anatomical nerve biopsy. When combined with the stimulating electrode connected to the needle into the needle up to a certain depth, the emergence and pulse current synchronous external anal sphincter contraction, indicating that the needle close to or reach the fourth recommendation nerve, then the pulse current turn to 0.5mA, external anal sphincter contraction is still that recommendation has been stabbed in the fourth nerve. Recording electrode piercing position, direction, depth. Adjust stimulated current parameter is set to square wave, frequency 1Hz, pulse width 50us, strength 5mA. Record anal sphincter EMG graphics. 5, perineural injection of drugs in the maintenance and monitoring of the current to stimulate the anal canal pressure changes in circumstances, in good positioning nerve at the fourth recommendation, Withdrawing without blood, nerves on both sides at the fourth recommendation was slowly injected drugs. A group of saline 0.5ml; B, C group was injected with 50g / L ropivacaine 0.1ml/kg epinephrine (1:250000); D, E group were injected with 0.5ml of ethanol volume fraction of 0.99 epinephrine (1:250,000) . Divided equally on both sides of the fourth recommendation nerve injections. 6, the measured 4 weeks after surgery under anesthesia in group B, D ARP anesthetized animals and fixed animals, and sacral hair removal around the anus, anal manometry bladders built into the connection tee, pressure calibration table, the pressure signal converters, sixteen physiological signal acquisition system PowerLab 16/30 (ML880), Dual BioAmp / Stimulator (ML408), measured under anesthesia animal ARP. 7, for 4 weeks after surgery under anesthesia in group B, D animals OK anal sphincter EMG examination from the puncture site in the more distant one indifferent electrode placement, and pulsed with needle connected to form a loop in the external anal sphincter SRE two sensing electrodes. Will be open to the stimulation current 1.5 mA, pulse frequency of 1 Hz. Press the recording electrode piercing position, direction, depth piercing, observe the movement of the external anal sphincter, when necessary, the current stimulus intensity was increased to 5mA. Record anal sphincter EMG graphics. 8, the measured 8 weeks after surgery under anesthesia in group A, C group, E group animals anesthetized and fixed ARP animals around the anus and sacral hair removal, anal manometry bladders built into the connection tee, pressure calibration tables, pressure signal converter, sixteen physiological signal acquisition system PowerLab 16/30 (ML880), Dual BioAmp / Stimulator (ML408), test animals under anesthesia ARP. 9, 8 weeks after surgery under anesthesia in group A, C group, E group animals OK anal sphincter EMG examination from the puncture site in the more distant one indifferent electrode placement, and pulsed with needle connected to form a loop in the external anal sphincter SRE two sensing electrodes. Will be open to the stimulation current 1.5 mA, pulse frequency of 1 Hz. Press the recording electrode piercing position, direction, depth piercing, observe the movement of the external anal sphincter, when necessary, the current stimulus intensity was increased to 5mA. Record anal sphincter EMG graphics. 10, specimen acquisition and processing were sacrificed after 4 weeks in group B, D group animals were sacrificed after 8 weeks of group A, C group, E group of animals, dissecting the external anal sphincter, injection site 4 recommended nerve root and nerve surrounding tissue, carefully observe the general morphology. Take the external anal sphincter, perineural tissue in 4% formaldehyde solution-fixed, paraffin-embedded, slice thickness 10um, HE staining, was observed under an optical microscope, the first four recommended drug injection sites around the nerve tissue, the external anal sphincter morphology changed. Take the fourth injection site recommended nerves, about the size of 1.0mm × 1.0mm × 1.0mm, immediately put to 4 ℃ precooled 30g ╱ L glutaraldehyde fixation, and then placed at 4 ℃, sequential ethanol dehydration, epoxy resin embedding, slice thickness 60nm, lead uranium stained transmission electron microscope at 4 recommended nerve root cell ultrastructural changes. 11, the data read and analyze the anal canal and anal sphincter pressure measured EMG findings through LabChart Reader V6.1 read and analyze 12, statistical analysis of each group before surgery, after surgery and after surgery ARP ARP difference to x ± s form, application SPSS 13.0 statistical software for statistical analysis, each group before surgery, after surgery ARP analysis of variance was used to compare differences between groups using LSD multiple comparison test, α = 0.05 as significant difference level. Each group of animals with bilateral external anal sphincter innervation after nerve EMG EMG check whether the apparent change in count data form, the application of SPSS 13.0 statistical software for statistical analysis, EMG animals in each group was used to compare the rate of change in apparent disorder bidirectional R × C table data Fisher's exact test. Results 1 animal survival in a group B animals died immediately after the operation, and the remaining animals survived to the end of the experiment, was to obtain specimens were executed. Animal postoperative survival rate is about 97%. 2, ARP, and changes in the external anal sphincter EMG used in this experiment before and after surgery and nerve EMG ARP to measure changes in the modeling results. Five groups of animals before surgery ARP was no significant difference (P> 0.05), the difference was statistically significant postoperative ARP (P <0.05), before and after surgery dARP difference was statistically significant (P <0.05), the experimental group and control group dARP differences were statistically significant (P <0.05), the experimental group dARP were higher than the control group, the experimental group dARP no significant difference (P> 0.05). Animals in each group before surgery and post-operative control animals anal sphincter EMG Tip No. 4 recommended neurologically normal; experimental animals after surgery, external anal sphincter EMG findings Tip: five groups of animals EMG changes after surgery was statistically significant difference in the rate of Significance (bilateral P <0.05), infer each group of animals with bilateral innervation external anal sphincter EMG EMG examination significant differences in the rate changed significantly, each group of animals left EMG changes were evident in group A 0%, B group 100 %, C group D group were 83.3%, E group, 67.7%, right EMG changes were evident in group A 0%, B group 100%, C group, D group, E group were 83.3%. 3, histology group D, E groups perineural tissue fiber content increased significantly, few inflammatory cell infiltration; A group and B, C group showed no abnormal changes. All the animals showed no obvious external anal sphincter pathology. 4, the nerve fiber cell ultrastructure scanning electron microscopy showed that the experimental group axonal degeneration, myelin lamellar structure disorder, vacuolization; controls axonal and myelin ultrastructure normal conclusion one. Incontinence animal model building local injection method is effective neurotoxic drugs, selective destruction of the external anal sphincter innervation, incontinence can be successfully fabricated animal models; 2 animal models of stability and good postoperative incontinence 8W, the experimental group still meets the diagnostic criteria for incontinence , combined with pathological findings, incontinence animal model stability; 3 simple, easy to popularize the subject chosen experimental laboratory reagents and equipment are common reagents and equipment, is easy to promote. The neural positioning, accurate and complete destruction dominate the external anal sphincter nerve trunk, quickly make the external anal sphincter paralysis, incontinence animal models to achieve the purpose of construction, operation is simple; 4 This concept of minimally invasive kinds of new animal model construction method in experimental animals trauma, animal mortality is low. The concept of minimally invasive animal ethics meet the requirement to treat experimental animals has important application value, after all, an animal model modeling method incontinence progress.
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