Establishing a rat model of spastic cerebral palsy by targeted ethanol injection

NEURAL REGENERATION RESEARCHVolume 8, Issue 34, December 2013www.nrronline.orgdoi:10.39/jissn. 1673-5374.2013.34.010 hpt/ww/rrolineorg; htp:ww.wzsyj.org]Yu YD, LiL, Shao XZ, Tian FT, Sun QL. Establishing a rat model of spastic cerebral palsy by targeted ethano/ injection. Neural RegenRes. 2013;8(34):3255-3262.Establishing a rat model of spastic cerebral palsy bytargeted ethanol injectionYadong Yu, Liang Li, Xinzhong Shao, Fangtao Tian, Qinglu SunDepartment of Hand Surgery, the Third Hospital of Hebei Medical University, Shiiazhuang 050051, Hebei Province, ChinaResearch HighlightsCorresponding author:(1) Targeted ethanol injection is a rapid and easy method of selectively damaging the pyramidalYadong Yu, M.D.. Chiefphysician, Departmentoftract for establishing a rat model of spastic cerebral palsy with gross morphological and pathologicalHospital of Hebei Medicalfeatures typical of the disease.University, Sihijazhuang(2) Ethanol, as a chemical ablation agent for complete pyramidal tract damage in the rat brain, has050051, Hebei Province,less impact on other parts of the brain. Thus, the characteritic symptoms and signs of the diseaseChina, yuyadong123@yahoo.cn.can be observed for an extended period of time in the model.Received: 2013-08-04AbstractAccepted: 2013-11-26Spastic cerebral palsy is generally considered to result from cerebral cortical or pyramidal tract(N20120504006)damage. Here, we precisely targeted the left pyramidal tract of 2-month-old Sprague-Dawley rats Author contributions: Yuplaced on a stereotaxic instrument under intraperitoneal anesthesia. Based on the rat brain ste-YD was responsible ftor studyreotaxic map, a 1-mm hole was made 10 mm posterion to bregma and 0.8 mm left of sagital suture.conception and drating ofA microsyringe was inserted perpendicularly to the surface of the brain to a depth of 9.7 mm, and 15performed the experiments.μL of ethanol was slowly injected to establish a rat model of spastic cerebral palsy. After modeling,Li L performed statisticalthe rats appeared to have necrotic voids in the pyramidal tract and exhibited typical signs andanalysis. Tan FT and Sun QLsymptoms of flexion spasms that lasted for a long period of time. These findings indicate that this isretrieval and experimentalan effective and easy method of establishing a rat model of spastic cerebral palsy with good re-implementation. All authorsapproved the final version ofproducibility. Ethanol as a chemical ablation agent specifically and thoroughly damages the pyra- the manuscript.midal tract, and therefore, the animals display flexion spasms, which are a typical symptom of theConflict of interest: Nonedisease.declared.Key WordsEthical approval: The studywas approved by theneural regeneration; brain injury; spastic cerebral palsy; animal models; ethanol; pyramidal tract; Experimental Animal Centerstereotaxic instrument; targeted injection; modeling methods; neuroregenerationChina and the ExperimentalAnimal Center of the ThirdUniversity, China.rebral palsy, and 40- -100%。of preterm baINTRODUCTIONbies and babies with low birth weight arefound to have cerebral palsy at birthl2!. InCerebral palsy is a syndrome caused bydeveloped countries, the rate of cerebralnon-progressivebrainnjuryandpalsy is 1-2.5%o. In recent years, neonatalmental defects during pregnancy, duringmortality and the stillbirth rate have de-childbirth or after birth up to infancy. Its maincreased significantly with the developmentmanifestations include motor dysfunctionof obstetric techniques and, nenpatal mori_中国煤化工and abnormal posture. Surveillance (cine; however, the inciderCerebral Palsy in Europe has shown thatpalsy is gradually increasinYHCNMH G2- -3%。of newborns per year suffer from rebral palsy is the most common type of3255Yu YD, et al. / Neural Regeneration Research. 2013;8(34):3255- 3262.Author statements: Thecerebral palsy, accounting for 45- 60% (neal injection of lipopolysaccharide inducesmanuscript is original, hascerebral palsy patients. Generally, spasticlocalized endotoxin-mediated damage to thenot under consideration bycerebral palsy is considered to be caused bywhite matter, which is closely related to theanother publication, has notcerebral cortical or pyramidal tract damage.local concentration of the lipoglycan in thebeen previously published inany language or any form,Patients with spastic cerebral palsy usuallytissuel32!. Neonatal ischemia-hypoxia cancontainsno disclosureofappear to have increased muscle tone,cause cerebral palsy in neonatal rats, withconfidential information orhyperreflexia, pathological reflex and otherneuropathological features similar to huauthorship/patent applicationsigns, with varying degrees of limb paraly-manslBs. Another study showed that intra-disputations.sis'5l. The pathogenesis of cerebral palsy isuterine injection of mucopolysaccharides viaSupplementarystill unclear, and there is still no effectivethe vagina, performed in rats at 15 days ofinformation: Supplementarydata associated with thistreatmentl. 681. Therefore, the clinical treat-pregnancy, produces behavior changes inarticle can be found, in thement of cerebral palsy is in need of signifi-the neonatal rats 1-21 days after birth!3 4.online version, by visitingwww.nronline. org.cant advancement. The establishment of aSome studies showed that neonatal ratsstable and reliable animal model has becomewere subject to intrauterine injection of mu-an important aim in the study of spastic ce-copolysaccharides, and then placed in arebral palsy9-12].hypoxic environment to establish a cerebralpalsy model. In addition, prenatal and peri-There are numerous studies on the use ofnatal hypoxia-ischemia is used by manyanimal models of spastic cerebral palsy.scholars to establish a rat model of cerebralThese include models generated by infection,palsy35. Currently, the most common meischemia and hypoxia, and bilirubin-inducedthod of establishing a cerebral palsy modelbrain injuy!13-20]. Sun and Li21] successfullyis subjecting neonatal rats to unilateral caro-prepared a rabbit model of cerebral palsytid artery ligation, followed by placement insimilar to human kernicterus by intraperito-n anoxic environmenti. A combinationneal injection of 300 mg/kg bilirubin. Mallardmethod is used by some researchers foret al 22 performed unilateral uterine arteryestablishing cerebral palsy models. Fcligation in pigs at 30 days of pregnancy toexample，Girard and colleagues'37] com-prepare a pig model of intrauterine ischemiabined mucopolysaccharide injection withand hypoxia. Zhang and colleaguesl3 re-carotid artery ligation in pregnant rats, whichmoved part of the cerebral cortex and me-ere placed in a hypoxic environmentdulla via a skull opening to generate a modelpostoperatively to create the cerebral palsyin rats. Dul24] exposed the motor cortex andmodel.inserted a microsyringe to a depth of 2 mmfor injection of 10 μL ethanol to damage theThe inflammatory response during cerebralmotor cortex. Dixon et al 1251 rapidly injectedhypoxic-ischemic reperfusion promotessaline into the rat brain to increase intra-secondary brain damage, which is one ofcranial pressure and damage brain tissue.the main causes of hypoxic-ischemic irWul26] and Yul27] established a model of spas-jury[38-40]. Interleukin-1B, a cytokine found intic cerebral palsy by electrical pyramidal tractthe early stage of hypoxic-ischemic braindamage. Studies from Delcourl28] and Vot-injury, is a critical signaling molecule in thetier!29] showed that prenatal ischemia cannerve-endocrine-immune system41. Zhangcause white matter injury, thereby leading toet al 1421 found that interleukin-1β is involvedcerebral palsy symptoms in rats. Repeatedin the pathogenesis of brain injury in adultintraperitoneal or intrauterine injection (rats during cerebral ischemia- reperfusiormucopolysaccharides into pregnant rats canIntrauterine infection in pregnant rats canstimulate the production of inflammatory cy-increase the mRNA levels of interleukin-1βtokines that damage oligodendrocytes, pro-and tumor necrosis factor in the brain ofmote the synthesis of other cytokines, andneonatal rats in a dose-dependent manner.increase nitric oxide synthesis, neutrophil" 中国煤化工. fbillary acidicinfiltration, and adhesion molecule expres-prbocampus andsion, thereby inducing white matter lesions inccTYHC N M H Gof astrocytesthe immature brain tissuel30-31. Intraperito-increases. The levels of myelin basic protein3256Yu YD, et al. / Neural Regeneration Research. 2013;8(34):3255- 3262.decrease and oligodendrocyte activity is alteredl43. In-flammatory cytokines can cause white matter lesions inthe immature brain by stimulating other cytokines, and bypromoting nitric oxide synthesis, neutrophil infiltrationand adhesion molecule expression, as well as by kllingoligodendrocytes44. Derrick et al (45) established aintrauterine ischemia and hypoxia model in pregnantrabbits to simulate birth injuries caused by placental ab-ruption. However, atypical symptoms or symptoms thatFigure 1 Rat behavior 72 hours postoperation.are of limited duration in these animal models produced(A) Spasms of the right limbs were visible in the modelby the above methods limit the in-depth study of spasticgroup. (B) No abnormalities were found in the limbs of ratscerebral palsy. Therefore, we precisely targeted the py-in the control group.ramidal tract using a stereotaxic instrument and used amicrosyringe for ethanol injection, aiming to establish aIn the control group, the rats had decreased activity andnew rat model of spastic cerebral palsy.ood intake, and poor mental status, but without flexionspasm of the right limbs, within 18 hours after surgery.Muscle tension was similar for the bilateral limbs, andRESULTStheir functioning was normal. After 36 hours, mentalstatus, activities, and food intake were becoming normal.Quantitative analysis of experimental animalsThe rats in the control group completely recovered theirTwelve male Sprague- Dawley rats were randomly as-activities, food intake and mental status 72 hours post-signed into model and control groups, with six rats in eachoperatively (Figure 1B).group. Rats in the model group were given an intracranialinjection of ethanol, targeting the left pyramidal tract, toTargeted injection of ethanol produced a localizedestablish a model of spastic cerebral palsy. In the controlpyramidal tract lesion in Sprague-Dawley ratsgroup, only a microsyringe was inserted into the brain. At Hematoxylin-eosin staining showed that at 72 hoursthe end of observation, one rat died from anesthesiapostoperatively, the rats in the model group had necroticoverdose in each of the two groups. In the end, five ratsvoids in the pyramidal tract, but without injuries to otherfrom each group were included in the final analysis.brain regions. In the control group, the pyramidal tractwas normal (Figure 2).Targeted injection of ethanol effectively established amodel of spastic cerebral palsy in Sprague-Dawley ratsBy 18 hours after modeling, rats in the model group hadno activity or food intake, and exhibited listlessness, ob-vious flexion spasms of the right forepaw and hind limbs(supplementary Video 1 online), and increased muscletension in the right upper and lower limbs. At 36 hourspostoperatively, the rats had a small amount of activefood intake and movement, and mental status also im-Figure 2 Morphology of the pyramidal tract 72 hours afterproved. During exercise, claudication of the right limbssurgery (hematoxylin-eosin staining, optical microscope,x 40)was apparent, and active and passive activities were(A) Rats in the model group appeared to have necroticaccompanied with a clockwise circling movement with avoids in the pyramidal tract. (B) No abnormalities werediameter of about 20 cm (supplementary Video 2 online),found in the pyramidal tract of control rats.and flexion spasm of the right limbs persisted. After 48hours, the diet in the model group was slightly improved,but still worse than in the control group. Greater activitywas displayed by the model rats, and mental status con-DISCUSSIONtinued to improve; however, flexion spasms still persisted.By 72 hours after modeling, the diet in the model rats hadAdvantages ar中国煤化宝3ted ethanolbegun to normalize, and mental status and physical ac-injection for esspastictivity were nearly normal, and flexion spasms of the rightcerebral palsyMYHCNM HGlimbs had stabilized (Figure 1A).The stereotactic technology used in the present study3257Yu YD, et al. / Neural Regeneration Research. 2013;8(34):3255- 3262.provides for more accurate positioning, thereby mini-In our experiment, the microsyringe was vertically in-mizing surgical errors and significantly improving theserted into the pyramidal tract with no damage to otherreproducibility of the experiment. Neonatal and pregnantparts of the brain. Postoperative signs of flexion spasmsrats have been used to establish models of cerebralwere very obvious, but there were other symptoms aspalsy. However, confounding factors can influence thevell. The control group was established to verify thatpathophysiology in neonatal and pregnant rats. Addi-ethanol is the prime agent destroying the pyramidal tract,tionally, neonatal and pregnant rats are much less re-rather than the microsyringe needle. In addition, Wsistant and tolerant to surgery than adult rats. Moreover,conducted preliminary experiments with many reagents,adult rats are inexpensive, easy to feed, and easy to testsuch as normal saline and hypertonic saline, to deter-behaviorally. In particular, the large brain volume in adultnine whether ethanol destroys the pyramidal tract byrats facilitates pathological and biochemical analysis ofsimply occupying volume. However, we found that therethe brain tissues4sl. Therefore, adult rats were preferredwere no signs of flexion spasm in adult rats administeredover neonatal and pregnant rats in the present study.normal saline, hypertonic saline or other liquid reagentsEthanol, a chemical ablation agent used in our study, is aafter recovery from anesthesia, indicating that liquidbetter alternative to electrical stimulation to destroy thereagents induce no substantial damage to the brain.pyramidal tract. The brain is the body's nerve center, andMoreover, the injected ethanol could also be absorbednerve impulse conduction is an electrochemical proafter destroying the pyramidal tract. We also attemptedcess7. Although the electric current is only dischargedto destroy the pyramidal tract using heavy metals suchfrom the tip of the insulated needle, the current conduc-as mercury, but the rats died postoperatively before re-tion path when discharging is uncontrolled. Although thecovery from anesthesia. After the injection of ethanol, thecurrent damages the pyramidal tract, we cannot controldrilling hole was sealed with wax, which not only effec-or monitor its impact on the other parts of the brain. Intively stopped the bleeding, but also prevented bloodcomparison, ethanol destroys the pyramidal tract com-from entering the drilling hole and prevented normal sa-pletely without damaging the other parts of the brain,line from entering the needle tract during postoperativethereby helping to maintain the specific signs andrinsing, helping to avoid dilution of the ethanol. It is verysymptoms of spastic cerebral palsy for a long period ofimportant to rigorously conduct the experiments, espe-time. The chemical damage by ethanol is fast and direct,cially in studies on dose-effect and time- dependent rela-and requires no complex operations or cumbersometionships. Furthermore, the needle insertion sites andexperimental procedures. Previous studies mostly foinjection zones were positioned in reference to The Ratcused on methodology or examination of microscopicBrain in Stereotaxic Coordinates (3'd edition), preciselymorphological changes, and some scholars emphasizedusing a stereotaxic instrument, thereby ensuring target-the histological changes. Spiegler et al [48] occluded fouring accuracy during injection.uterine arteries in maternal rats at 18 weeks of preg-nancy for 45 minutes, and then collected the brain tissuePrecautions during targeted injection of ethanol forof neonatal rats for histological examination. Periventri-establishment of the rat model of spastic cerebralcular leukomalacia is the major pathological feature ofpalsyneonatal hypoxic-ischemic brain damage, and is theWe should closely observe the rat's breathing and heartmain cause of cerebral palsyl49. Numerous experimentalbeat throughout the experimental procedures, includingstudies have focused on periventricular leukomalacia,anesthesia. A 20 mL syringe and a urinary catheter areoligodendrocyte loss and glial cell proliferationl50-51. Rid-necessary for suctioning to avoid suffocation in rats indle et al [52] observed changes to the sheep brain aftercase there are signs of airway obstruction'5s. Inaccurateacute hypoxia and ischemia reperfusion by measuringthree-dimensional coordinates could make the microsy-the blood flow distribution, oxygen saturation and patho-ringe needle deviate from the target site, thereby result-logical indexes in fetal sheep brain tissue, and found thating in a failed experiment or unsatisfactory experimentalperiventricular white matter injury in the animal modelresults. In addition, stable skull fixation, without penetra-was pathologically similar to white matter lesions in pre-tion of the bilateral tympanic membranes, is required.term infants. In a study by Wang et al5s, 5-day-oldHowever, this was relatively poor in our experiment.postnatal rats underwent intracranial injection ofTherefare, researchers should be careful and gentle3-nitropropionic acid. The pathological changes in theduring the oper中国煤化工d isocation ofbrain tissues included widespread damage to the whitethe skull. In paH:during the tar-matter and cerebral cortex, corpus callosum atrophy andgeted injectionc N M H Gimental erors,ventricular dilatation.but will also damage the microsyringe, thus causing ex-3258Yu YD, et al. / Neural Regeneration Research. 2013;8(34):3255- 3262.perimental failure. For the three-dimensional coordinates,spastic cerebral palsy. The method is rapid and easy,the target site should not be selected 0.7 mm anterior toand produces a model with manifestations, pathologicalthe line between the ears and 1 mm to the midline, so aschanges and symptoms typical of cerebral palsy.not to damage the sinus and cause bleeding. However,we inserted the microsyringe 1 mm distal to the sagittalsuture, which resulted in more bleeding during the expe-MATERIALS AND METHODSriment. In this experiment, we had to carry out the tar-geted injection of ethanol after hemostasis.DesignA randomized, controlled, animal experiment.The rat model of spastic cerebral palsy prepared usingthe method described in this study exhibits obvious signsTime and settingof limb spasms, which continue to manifest for an ex-All experiments were performed at the Experimentaltended period of time. The operating procedure is simpleAnimal Center, the Third Hospital of Hebei Medical Uni-and standardized with perfect repeatability and accurateversity, China from September 2010 to March 2012.positioning. The experimental results are described qua-litively. Further studies are required to determine theMaterialsoptimal ethanol dose and the dose-effect and time- effectA total of 12 male Sprague-Dawley rats of specific patho-relationships.gen-free grade, aged 2 months and weighing 250士12 gwere provided by the Experimental Animal Center, theCharacteristics and application of the targetedThird Hospital of Hebei Medical University, China (crtifi-injection of ethanol for establishment of the ratcate No. SYXK (Ji) 2008-0026). The rats were housed in amodel of spastic cerebral palsyfeeding room, specific pathogen-free grade, at 22土1°C,The animal model described in the present study shows50-70% humidity, with a 12-hour lightdark cycle (llumina-obvious symptoms of flexion spasms and typical signstion: 150- -200 |x). All experimental procedures were inthat endure for a long term. The modeling method isaccordance with the Guidance Suggestions for the Caresimple and standardized with good repeatability. Basedand Use of Laboratory Animals, published by the Ministryon this model, we can carry out in-depth studies of theof Science and Technology of Chinal55.pathology of cerebral palsy and assess treatment strate-gies for the disease. For example, the model can allowMethodsus to examine the changes in the central nervous systemAnimal anesthesiaand peripheral nerves during cerebral palsy. We can alsoThe rats were anesthetized by intraperitoneal injection ofinvestigate whether end-to-end anastomosis of the10% chloral hydrate (0.004- -0.005 mL/g). Then, Wenerves that control the flexor and extensor muscles canclosely observed the vital signs of anesthetized rats. Thealleviate the flexion spasms. Using this model, we carrats were anesthetized successtully if they exhibited thealso investigate whether nerve growth factor treatment orfollowing: deeper and slower breathing, limb and musclepartial transplantation of the pyramidal tract can accele-weakness, delayed corneal reflex, and no response torate the disappearance of flexion spasms when the mostsharp stimulation of the tail6l.typical symptoms appear, allowing researchers to ex-plore new approaches and methods for the treatment ofFixation of ratscerebral palsy and other brain injuries. In addition, whileFirst, the rats were placed in a stereotactic apparatusthe symptoms are maintained for an extended period in(Anhui Zhenghua Biologic Apparatus Facilities Co., Ltd.,this model, they gradually disappear. Therefore, we canHuaibei, Anhui Province, China) with the root of the incisorexplore the mechanisms underlying recovery from thehooked firmly with the incisor fixator. Then, the left andflexion spasms. Such studies may provide insight thatright ear bars were inserted into the rat ears, followed bywill help accelerate recovery from cerebral palsy.appropriate pressure until the rats appeared to haveslightly protruding eyes, and we tightened the knob of theAlthough there has been significant progress in our un-ear bars. We adjusted the incisor to align the bregma withderstanding of disease pathogenesis and animal modelthe lambdal51. When the incisor bar was located at 3.9土preparation, it is necessary to identify better indicators of0.5 mm below tl中国煤化工t skull was in thethe disease and to generate novel animal models thathorizontal positil:.he skull duringbetter simulate spastic cerebral palsys4. In this study, wesurgery, we cheYHCNMHGknobwastigh-targeted the pyramidal tract to establish a rat model of tened. After checking, the rat head should be stably and3259Yu YD, et al. / Neural Regeneration Research. 2013;8(34):3255- 3262.firmly fixed on the stereotactic apparatus.mental state, and spasticity in rats at 18, 36, 48 and72 hours postoperatively.Preoperative preparationThe rat skull was exposed and disinfected with iodine inalcohol and ethanol. The microsyringe (Zhenhai GlassABInstrument Factory, Ningbo, Zhejiang Province, China)containing ethanol (Shanghai Nanxiang Reagent Co.Ltd., Shanghai, China) was fixed firmly in the stereotacticapparatus.CPreparing the model of spastic cerebral palsyA parietal incision about 2-cm-long was made over themidline through the skin, subcutaneous tissue, deep fasciaand periosteum, layer by layer, with the cut skin fixed onthe stereotactic apparatus. Ater removal of the perios-teum, the bregma and sagittal suture were exposed (Figure 3A). Based on The Rat Brain in Stereotaxic Coordi-nates (3"d edition)5, the stereotaxic coordinates weredetermined: a hole, about 1 mm in diameter, was drilled10 mm posterior to bregma, 0.8 left of sagittal suture, and9.7 mm ventral to dura (Figure 3B). Then, we determinedFigure 3 Process of preparing a rat model of spasticwhether there were deviations from the stereotaxic coor-cerebral palsy.dinates (Figure 3C). If bleeding, cotton balls with saline(A) Expose the bregma and sagittal suture. (B) Accuratewere used for hemostasis by compression rather thanorientation. (C) Drill the parietal bone. (D) Insert therepeated rubbing. The drilling was stopped when the bitmicrosyringe into the skull. (。the skull. (E) Sew up the incision.reached the dura mater. A 5 mL syringe needle was usedto determine whether the hole was drilled through the skullPathological observation of the pyramidal tract usingto avoid damage to the microsyringe needle during inser-hematoxylin-eosin stainingtion. Then, the microsyringe needle was verically insertedApproximately 72 hours after surgery, one rat was ran-into the cranium to a depth of 9.7 mm (Figure 3D) for in-domly selected from each of the two groups. After anes-tracranial injection of 15 μL ethanol. Afer injection, thethetization with 10% chloral hydrate (0.004-0.005 mLg),microsyringe was removed and brain cottons were usedthe rats were placed in a sterile operating table to openfor adequate hemostasis by compression. Afterwards, thethe parietal incision and clear the wound and subcuta-driled hole was sealed with bone wax and the wound wasneous hematoma. The original incision was extendedrinsed with normal saline and sewed (Figure 3E). In thefrom the tip of the rat nose to the neck to expose thecontrol group, the microsyringe was inserted into the brainentire skull clearly. The bone wax fling the drlled holewithout ethanol injection.was removed. A transverse incision was made using awire saw from the eyebrow area through the temporalPostoperative treatmentline to the external occipital protuberance, which was notThe rats were removed from the stereotactic apparatustoo deep. Then, the skull was opened along the trans-and placed in a 25- -28°C environment. Before awakening,verse incision to separate the bone marrow from thethe rats were monitored for general conditions. Generally,spinal canal at the foramen magnum using a small cut-the rats would recover from anesthesia at postoperativeting bit. A micro scissor was used to cut off the olfactory2 hours.bulb and optic nerve, and then, a knife was inserted intothe sella to isolate the pituitary gland.The symptoms and signs were compared between themodel and control groups within 24 hours postoperatively.Finally, the brain with the pituitary gland was taken out.Rats were only given feeding without postoperativeAfter removal of the dura mater, the brain was imdressing or antibiotic treatment.mersed in f中国煤化工_brain tissuel581After fixation, tdrated, clearedGross observationand immersedC N M H Gsue specimenDetailed observations were done for food intake, activity,was placed in melted paraffin wax until solidified. The3260Yu YD, et al. /Neural Regeneration Research.2013-83)3255-3262 .paraffin block was trimmed, placed on the wooden[13] Li J, Chen G Research of cerebral palsy animal models.holder and cut into slices, 5 um in thickness. 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