| 1. BackgroundAcute spinal cord injury (SCI) often occurs with vertebrae fracture and dislocations in clinic. Recently some studies reported that approximately17.75%to51.00%of SCI happened in the level of the cervical spine. Most clinical cases of SCI occurs at the C5level, followed by C4and C5. Most of models are rodents, whose exprimental methods including cutting, contusion, compressions and so on. They all did not well minic the mechanism of fracture dislocationsresulting in SCI in clinic. In2005, Fiford reported the characerization of the primary SCI due to the thoracic spine fracture-dislocations. In2009, Choo M founded up a cervical SCI model due to fracture-dislocations of the cervical spine. He and his colleagues made C4and C5fracture dislocated2.5mm to induce primary SCI, and then compared with contusion SCI. And then in2012, Shahrokni reported the model of C5/6fracture dislocation whose relative displacement was1.5mm, and focused on studying biomechanical stability of the rat cervical clamp made of PEEK, withoutdata of histology and behavior.Our previous study has demonstrated the C4/5relativelly dislocated1.90mm at speed of200mm/sec can induce cervical graded primary SCI. Thus the purpose of this study was aiming to study the characterization of primary and secondary cervical SCI. We conducted a preliminary analysis histological and biomechanical characteristionof cervical SCI, as well as histological and behavior characterizationof secondary SCI (8weeks). Based on the data of behavior and histopathology, we developed a reproducible two severities cervical SCI model. This characterized graded cervical injury model is presented as a new tool for the assessment of potential therapeutic interventions for SCI repair.2. Objective2.1To conduct two different cervical spine fracture and dislocation displacement, and establish a graded primary SCI modelin rats;2.2To characterizehistological and functional outcomes of the SCI model at different dislocated displacement in rats.3.Materials and MethodsForty-two male Sprague-Dawley rats were randomized to three groups:(1)1.65mm group;(2)1.80mm group;(3) surgical control group. The experiment was conducted on two time points:0weeks post-injury (primary spinal cord injury) and8weeks post-injury (primary spinal cord injury).3.1To study characterizations of graded cervical primary SCI;This section used eighteen adult male Sprague-Dawley (SD) rats (weight300g to335g).The animals were randomized to two groups the1.65mm group(n=6) and the1.80mm group(n=6),according to the displacement of fracture-dislocations. Six unjured animals served as control group. The C4/5bilateral facets were removed by milling drill. Therostral clamp holding notch of C2(1/2), C3, C4was connected to the frame of stereotaxic apparatus and kept stationary during the injury process, while the caudal clamp holding C5, C6, C7notchwas connected to a material testing machine. Then the caudal clamp drived dorsally up to1.65mm or1.80mm at a speed of200mm/s, stayed for0.3s and returned to the original position, to inducefracture dislocation between C4and C5. During the process, the machine had recorded the experimental parameters, including time, force and displacement. After injury, the cervical spinal cord contains C4-C5were harvested by perfusing paraformaldehyde. The process of control group was the same as the injuried group, excepting fractures-dislocations. These frozen sagittal slices were stained by Hematoxylin&Eosin (HE) to calculate the volume of hemorrhage of spinal cord in white matter, and gray matter independently.3.2To study characterizations of graded cervical secondary SCI;Behavioral testForelimb locomotor score (FLS) was performed on1day before surgery,1～8weeks (once a week) post-injury to evaluatethe forelimbs function by slow notion video playback.Grooming test was carried out on1day before surgery,8weeks post-operation (once a week).We induced spontaneous grooming by applying cool tapwater to the animal’s head and back, and videotaped to evaluate therange of forelimbs motion in rats.We focused on the evaluation of forelimbs’muscle strength changes post-injury by grip strength meter.We made the rats accustom to being held forl week pre-injury, and obtained the preoperative baselines on1day before injury. The strength of left and right forelimbs in rats was separately measured on day1,4,7,14,21,28,35,42,49,56post-operatively.Histology At week8post-operation, rats were euthanatized by an overdose of sodium pentobarbital euthanasia solution (80mg/kg), and then intranscardially perfused with0.01M phosphate-buffered saline, followed by4%paraformaldehyde. The segments from C3-7(7-mm-long) whose center is C4/5was removed by gentle disesction, dehydratied by gradientsucrose solution, and quick-freezed by dry ice and stored at-80℃.The spinal cord was cut into20-μm-thick transverse sections, with a400μm intersection interval. Every section was collected such that10sets of adjacent("sister") sections, each representing4000μm of tissue.The slices was collected at-20℃. Four sets slices were randomly taken to perform with HE stain to observe spinal cord gross morphology, myelin stain (luxol fast blue) to evaluated myelin loss, nissl stain to count motor neuron numbers, and GFAP immunohistochemistry stain to analysis glial response.4. Results4.1Graded primary SCI due to different displacement;In1.65mm group and1.80mm group, on average, the maximal force was (16.45±3.56) N and (15.57±5.73)N respectively, and actual maximum displacement was (1.64±0.03) mm and (1.80±0.02) mm. The speed of two groups was (199.4±1.7)mm/s and (201.5±2.0) mm/s, both closed to200mm/s.There was no significant difference in the maximal force between the two groups (Pforce=0.756). The actual maximum displacement got very close to the target displacement. The device produced no significant difference levels of speed between two experimental groups (Pspeed=0.084).The volume of hemorrhage in white matter were (0.01±0.02) mm3and (0.09±0.05)mm3, while in gray matte was (0.58±0.15) mm3and (0.88±0.21) mm3in the1.65mm group and the1.80mm group respectively. The volume of hemorrhage of gray matter and white matter between the two groups was significant different.4.2Behavioral and Histological analysis of graded spinal cord secondary injury Behavioral assessmentOn1day post injury, The1.65mm group averaged (0.8±0.4), and the1.80mm goup averaaged (0.2±0.4),which both represent slight movement of only one joint(usually is shoulder)(P=0.810). At1week post injury, the1.65mm group demonstrated a mean score of (3.0±1.9), which represented a moderate movement of two joints or a extensive movement of single joint. The1.80mm group averaged6.5±3.0, which represented extensive movement of two joints (shoulder, elbow) and a slight movement of joint (wrist). From2to8weeks post injury, all injured animals had progressive improvements in the forelimb locomotor scores. At three weeks post injury,the animals in1.65mm groups reacheda recovery platform, and then achievedlittle improvement. At3th weeks post injury, the1.65mm group and thel.80mm group scored (12.2±1.5)and (10.6±1.5) respectively. At endpoint, the forelimb locomotor scores were (14.3±0.7) and (12.1±0.9) in the1.65mm group and the1.80mm group. There was signicant difference between two groups at each time point.Animals with SCI, however, showed significant deficit at all time points post-injury. The injured groups both significant improved from day1to week1,and week2to week8. One week after injury animals in both groups demonstrated limitations in the ablity to imitiate a grooming sequence from behind the nose(1.65mm group,1.4±0.7;1.80mm group,1.1±0.8,P>0.05). Eight weeks post-operation, the maximal contact made by forlimbs of all injured animals demonstrated limitations behind the eyes(1.65mm group,3.4±0;1.80mm group,3.3±0.8,P>0.05). There was no significant overall preference for the scores of grooming test at all time points. The grip strength of three groups (1.65mm group,1.80mm group, control group)was (1.64±0.13) N,(1.59±0.10) N and (1.63±0.08N) respectively (P>0.05) before operation. At week1post injury, grip strength of the1.65mm group and the1.80mm group was (0.49±0.18)N, and(0.83±0.13)N (p<0.05),respectively,while that in the control group was (1.44±0.20)N. All injuried animals in the1.65mm group and the1.80mm group recovered over time. At experimental endpoint, there was significant difference in grip strength between the1.65mm group (1.55±0.24) N and the1.80mm group (1.39±0.16) N, and both less thanthe control group (1.87±0.13)N.We found no significant difference existed in left and right forelimb grip strength, which meaned this model was a well bilateral cervical SCI model.Histological analysisAt the endpoint of this study, anatomical damage of spinal cord was analysised by HE stain. The1.65mm group had larger spared issue at the lesion epicenter when compared to the1.80mm group. The area of spared spinal cord at lesion epicenter was (3.24±0.82) mm2VS (2.15±0.51) mm2. Measurements of sparied spinal cord tissue volume were taken at400-μm intervals through4mm of the cord centered on the lesion epicenter. The rostro-caudal volume of the spinal cord in the1.65mm group was(18.07±2.31)mm3, also larger than that in the1.80mm group (16.01±2.34)mm3, while the control group was (25.38±1.05)mm3. There was significant differenerence among the three groups. Based on these results, we demonstrated that the more displacement, the more severe SCI.At eighth weeks post-operation, a robust increase glial response was observed around the cystic cavity and in the slices adjacented to the epicenter in both gray and white matter of all injuried groups, while that was not similar status existed in the control group.When glial responseof spinal cord was analysised, all injuried groups showed a significant increase compared to unjuried control. Glial response in the1.80mm group was more robust than that in the1.65mm group.IOD value of myelin stain were taken at400-μm intervals through4mm of the cord(a total of11slices) centered on the lesion epicenter.There was significant difference in IOD value among three groups(P=0.000).The total IOD value was(2.87±0.34)x105in the1.65mm group,(1.14±0.33)x105in the1.80mm group, and (0.54±0.30)x105in the control group. Both injuried groups showed a significant myelin loss compared with the control group, and significant more myelinloss in the1.80mm group compared to the1.65mm group (P=0.027). The section of1600μm caudal to lesion epicenter showed that the1.65mm group had a much larger rim of spared tissue than the1.80mm group.There was only narrow rim of myelin remained in the1.80mm group. The myelin less tightly packed in the1.80mm group with more holes and less stain than the1.65mm group and the control group.In both injuried groups, motor neurons were lost at the lesion and rostro-caudally for2.0mm. This loss was significant when comparedto the control group (P=0.000;) and was most evident around the lesion epicenter(almost completely destroyed).The section of1600μm caudal to lesion epicenter showed that motor neurons were swollen, disrupted or shrunk.The numbers of motor neurons in the1.65mm group was larger than the1.80mm group(P=0.048).5. ConclusionsThis study successfully established a graded SCI model due to different fracture-dislocations of the cervical spine. We have initially confirmed that C4/5fracture-dislocations at displacement of1.65mm and1.80mm in rats can iniduce graded spinal secondary spinal cord with different histological and behavioural functional outcomes.This characterized cervical SCI model is presented as the model for studying SCI mechanism and the assessment of putative therapeutic interventions for SCI repair. This model allows the application of data and analysis toward the most relevant form of human spinal cord injury. |
PDF Full Text Request