ultrastructural scoring of graded acute spinal cord injury in the rat

UMEROUS substances have been tested in experi-mental neurotrauma models. Behavioral tests inwhich the results were difficult to interpret have

often been used as the main outcome measurement, with-out careful and quantitative histological examination. Theabsence of such data has made the testing of certainpromising drugs in clinical trials problematic.23 Many his-tological methods have been used to evaluate experimen-tal SCI to obtain information on injury-related pathophys-iology and the effects of neuroprotective agents on thespinal cord. Histological methods by which recovery fromexperimental SCI is assessed comprise posttraumatic cystmeasurements,6,12,18,27 axonal tracer–determined cell countsin the cerebrum and midbrain,9,19,20,22,26 cell counts in thepyramidal tract of the injured spinal cord,3,25 and nonquan-tified ultrastructural evaluation.1,13

A posttraumatic cystic cavity develops in the spinal cordof a rat subjected to severe spinal cord contusion.12 In stud-ies on neuroprotection, the maximum cross-sectional areaand the volume of the cavities have been estimated for

quantitative evaluation. In this evaluation, tissues are seri-ally sectioned; every 10th section of tissue is photographedand magnified; and measurements are performed using animage analysis system. Thus, numerical values can be usedfor statistical analysis.18

Axonal tracers such as HRP have been used in thequantitative evaluation of SCI. After HRP administration,coronal sections obtained in the cerebrum and midbrainare processed for HRP reactivity. Labeled corticospinaland rubrospinal neurons are counted to determine a corti-cal score and red nucleus score. The degree of preserva-tion of dorsal columns including the corticospinal tracts atthe injury site correlates with the cortical score, whereasthe red nucleus score is related to the degree of the preser-vation of the lateral columns.19 Fluorogold, an axonal trac-er, has also been demonstrated to label rubrospinal neu-rons after SCI. Rubrospinal neuron counts correlate wellwith functional scores.20

The concentration of axons distal to the injury site hasbeen shown to diminish markedly after acute spinal cordtransection or compression injury. Axons are counted insections after being stained with the Holmes stain.25 Thequantitative analysis of axons in acute SCI has also beenperformed ultrastructurally.3 In that study, investigators

J. Neurosurg: Spine / Volume 97 / July, 2002

J Neurosurg (Spine 1) 97:49–56, 2002

Ultrastructural scoring of graded acute spinal cord injury in the rat

ERKAN KAPTANOGLU, M.D., SELCUK PALAOGLU, M.D., PH.D., H. SELCUK SURUCU, M.D., PH.D.,MUTLU HAYRAN, M.D., M.S., AND ETEM BESKONAKLI, M.D., PH.D.

Department of Neurosurgery, Hacettepe University Institute of Neurological Sciences and Psychiatry,Spinal Research Laboratory, Ankara Numune Education and Research Hospital; and Departments of Neurosurgery and Anatomy, and Department of Preventive Oncology, Oncology Institute,Hacettepe University, Faculty of Medicine, Ankara, Turkey

Object. There is a need for an accurate quantitative histological technique that also provides information on neu-rons, axons, vascular endothelium, and subcellular organelles after spinal cord injury (SCI). In this paper the authorsdescribe an objective, quantifiable technique for determining the severity of SCI. The usefulness of ultrastructural scor-ing of acute SCI was assessed in a rat model of contusion injury.

Methods. Spinal cords underwent acute contusion injury by using varying weights to produce graded SCI. AdultWistar rats were divided into five groups. In the first group control animals underwent laminectomy only, after whichnontraumatized spinal cord samples were obtained 8 hours postsurgery. The weight-drop technique was used to pro-duce 10-, 25-, 50-, and 100-g/cm injuries. Spinal cord samples were also obtained in the different trauma groups 8hours after injury. Behavioral assessment and ultrastructural evaluation were performed in all groups.

When the intensity of the traumatic injury was increased, behavioral responses showed a decreasing trend. A simi-lar significant negative correlation was observed between trauma-related intensity and ultrastructural scores.

Conclusions. In the present study the authors characterize quantitative ultrastructural scoring of SCI in the acute,early postinjury period. Analysis of these results suggests that this method is useful in evaluating the degree of traumaand the effectiveness of pharmacotherapy in neuroprotection studies.

KEY WORDS • electron microscopy • scoring • spinal cord injury • ultrastructure

N

49

Abbreviations used in this paper: HRP = horseradish peroxi-dase; SCI = spinal cord injury; SEM = standard error of the mean; TEM = transmission electron microscopy.

used axon counting to evaluate axonal pathophysiologyfollowing trauma.

Although a variety of ultrastructural pathological fea-tures of SCI have been described previously, a quantitativeultrastructural assessment of the changes in the postinjuryspinal cord in conjunction with behavioral evaluation hasnot been performed. We previously reported for the firsttime that using electron microscopy to score SCI is veryuseful in neuroprotection studies.15,16 In the present studywe investigate the differential effects of graded SCI onspinal cord ultrastructure. Our goal was to determine if ul-trastructural scoring of acute SCI in the early posttraumat-ic period shows correlation with trauma-related intensityand clinical findings after weight-drop contusion injury.

Materials and MethodsAdult male Wistar rats, weighing 190 to 230 g, were used for the

study. The surgical procedure was performed after induction of gen-eral anesthesia (10 mg/kg xylazine and 60 mg/kg ketamine hydro-chloride intramuscularly). Rats were placed in the prone position.After making a T6–10 midline skin incision, the paravertebral mus-cles were dissected. The T7–9 spinous processes and laminar arcswere removed, and a laminectomy was performed. The meningeswere not disrupted. The weight-drop method2 was used to produce10-, 25-, 50-, and 100-g/cm spinal cord contusion injuries in thetrauma groups. The graded force was applied using stainless-steelrods (3-mm-diameter tip, weighing 2, 5, 10, and 20 g) by droppingvertically through a calibrated tube. Injury apparatus included a 5-cm guide tube that was positioned perpendicular to the center ofthe spinal cord. After surgical and traumatic interventions, silk su-tures were used to close the wound in layers.

Experimental Groups

The rats were randomly divided into five groups of 10 rats each;they were perfused and killed 8 hours postoperatively after undergo-ing clinical evaluation. In Group 1 rats (sham-operated controls) onlylaminectomy was performed, and a 1-mm-thick transverse section ofnontraumatized spinal cord tissue was obtained after clinical evalua-tion. The Groups 2 to 5 rats (trauma-induced animals) underwent sur-gical and traumatic interventions. In these trauma groups, rats weresubjected to 10-, 25-, 50-, and 100-g/cm impact injury, respectively.A 1-mm transverse section of traumatized spinal cord tissue was alsoobtained 8 hours postsurgery after clinical evaluation.

Functional Evaluation

Clinical behavior was assessed 8 hours after trauma by using theinclined-plane technique21 and the Basso-Beattie-Bresnahan scoringsystem.5 This scale includes 21 different levels of hind limb move-ments. Mean Basso-Beattie-Bresnahan scores of both legs were ex-amined. Observers undertook the evaluations in a blinded fashion.

Obtaining Samples From the Spinal Cord

Eight hours after surgery, following clinical evaluation, anesthe-sia was reinduced and animals underwent perfusion. To guaranteethat equal quantities of tissue were obtained in the different groups,samples were taken from the spinal cord as follows: after perform-ing T-7, T-8, and T-9 laminectomies, the area between the T-6 andT-10 laminar arcs was measured. At the level of T-8 trauma wasproduced in the middle of the area where laminectomy was per-formed. Additionally, a nonabsorbable No. 3-0 suture was insertedto paraverterbal muscles adjacent to lesion site to confirm the cen-ter of the traumatized area. A 1-mm cross-sectional area of spinalcord at the epicenter of trauma was removed. Samples for whitematter were obtained from the dorsal column of the spinal cordwhere the corticospinal tract is located, and for gray matter sampleswere taken from the anterior gray horns where motor neurons areprimarily located (Fig. 1).

Spinal cord samples were collected in randomly numbered con-

tainers, and given to the authors as each sample in its own numberedcontainer. After evaluating the numbered tissues by a person blindedto the study, the results were collected in the appropriate group lists.

Sample Preparation for Electron Microscopy

The tissues used for TEM were obtained after transcardiac perfu-sion with phosphate-buffered 2.5% glutaraldehyde/2% paraformal-dehyde solution. The tissue samples were kept in the perfusion solu-tion for 24 hours, postfixed with phosphate-buffered 2% OsO4 for 1hour and then dehydrated in a graded series of alcohol. After aral-dite embedding, 1- to 2-�m semithin sections were obtained usingan ultratome, stained with toluidine blue, and visualized using alight microscope. The same ultratome was used to obtain 60- to 90-nm-thick sections, which were contrast stained with uranyl acetateand lead citrate, and visualized using an electron microscope.

E. Kaptanoglu, et al.

50 J. Neurosurg: Spine / Volume 97 / July, 2002

FIG. 1. Drawing representing a transverse section of rat spinalcord. Letters in boxes indicate the areas in which samples wereobtained (W = white matter [corticospinal tract, ventral portion ofdorsal column; 50 axons/sample evaluated]; G = gray matter [ante-rior motor neurons; 20 neurons/sample were evaluated]).

TABLE 1Grading system for quantitative evaluation

of ultrastructural findings

Category Score

axonal myelinnormal myelin layers 0vesiculated myelin 1cracked myelin layers 2honeycomb & extruded vesicles 3

general axonalnormal 0light edema 1mild edema 2severe edema & loss of structure 3

intracytoplasmic edemaabsent 0light 1mild 2severe (cell membrane defect) 3

nucleusnormal 0clumping 1sparse chromatin 2severe damage 3

vascular endotheliumnormal 0light edema 1mild edema 2severe edema 3

Samples were evaluated according to the following grading system.

Electron Microscopic Evaluation Grading System

The grading system, used for quantitative evaluation, was basedsimilarly on the principles of the methods used for evaluating different tissue samples17,24 and the spinal cord15,16 (Table 1). Fif-ty axons, 20 neurons, and 10 capillaries for each sample were eval-uated. In each group, axonal degeneration, axon–myelin split-ting, intracytoplasmic edema, nuclear damage, and vascular endo-thelial damage were evaluated, counted, and presented as meanscores � SEMs.

Statistical Analysis

Statistical analysis was performed using one-way analysis of vari-ance and the Bonferroni t-test. Data are expressed as the means �SEMs in Table 2. A probability value less than 0.05 is consideredstatistically significant.

The correlation between the clinical functional scores and thetrauma intensity was analyzed using the Spearman nonparametriccorrelation test. The trauma group TEM scores were analyzed usingthe nonparametric test for trends across ordered groups.8

Results

Functional Findings

Inclined-plane scores obtained in the laminectomy-onlygroup were higher than in all other groups. There was adecreasing trend in inclined plane scores with increasingtrauma intensity. There was no significant difference be-tween the 25- and 50-g/cm-injured groups (p � 0.05). Allother groups showed statistically significant differences(p � 0.05). The Basso-Beattie-Bresnahan scores alsoshowed a similar trend. Laminectomy-only rats showedthe highest scores; the scores decreased with increasingtrauma intensity. All groups showed statistically signifi-cant differences (p � 0.05), except the 25- and 50-g/cmtrauma groups (p � 0.05).

Ultrastructural Findings

In the control (laminectomy-only) group, the spinalcord gray and white matter was normal and intact (Fig. 2).

In the 10-g/cm weight-drop group, the neuronal nucleiappeared almost intact, although some marginalization ofthe chromatin material was observed. The cytoplasm was


Ultrastructural scoring of experimental SCI

51

TABLE 2Clinical functionality scores according to intensity of SCI*

Percentiles

Score† Intensity (g/cm) Means � SEMs 25 50 75

degree of inclined plane 0 68.33 � 1.05 65.00 70.00 70.0010 52.50 � 2.14 48.75 52.50 56.2525 42.50 � 2.14 38.75 42.50 46.2550 42.50 � 2.14 38.75 42.50 46.25

100 35.83 � 2.39 32.50 37.50 40.00Basso-Beattie-Bresnahan score 0 19.83 � 0.17 19.75 20.00 20.00

10 13.83 � 0.75 12.00 13.50 16.0025 9.83 � 0.70 8.75 9.50 10.7550 8.67 � 1.78 6.25 9.00 11.75

100 2.67 � 0.67 1.00 2.50 4.25

* Ten samples were evaluated for each intesity level of SCI.† p � 0.001 (Spearman nonparametric correlation test).

FIG. 2. Electron microscopic studies. Upper: Gray matter ob-tained in control (laminectomy-only) group, showing the normalappearance of nucleus (N), nuclear membranes (Nm), and axons(A). Lower: White matter of control (laminectomy-only) groupdemonstrating the normal appearance of axon, myelin sheath (S),mitochondria (M), and neurofilaments (Nf). Original magnifica-tion � 6000, bar = 1 �m.

less condensed with fewer organelles than normal (Fig. 3upper). The white matter was more affected by the trau-ma. The axons showed some dispersion in the myelin lam-ellae and edema in the mitochondria. The vascular endo-thelium was intact and the capillary lumina were patent(Fig. 3 lower).

In the 25-g/cm weight-drop group, the gray matter wasalso affected. There were numerous neuronal bodies withsparse chromatin and few organelles in the cytoplasm, in-cluding some swollen mitochondria (Fig. 4 upper). Thedamage in the white matter also increased with edematousspaces inside the axons, cracked myelin layers, and edema-tous areas in the periaxonal cytoplasm. The endothelialcells of the capillaries showed some swelling with narrow-ing of the lumen (Fig. 4 lower).

In the 50-g/cm weight-drop group, the nuclear damageadvanced with large empty spaces in the cytoplasm (Fig.5 upper). In the white matter, the myelin layers of the ax-ons were nearly destroyed with edematous areas inbetween (Fig. 5 lower).

In the 100-g/cm weight-drop group, the neuronal nucleishowed severe damage and the cytoplasm was composed

of a large empty space with no organelles. The vascularendothelium was swollen, and the lumen was completelyblocked (Fig. 6 upper). In the white matter, there were fewaxons, large edematous spaces, and some erythrocytes dueto bleeding (Fig. 6 lower).

Correlation of Behavioral and Ultrastructural Results

The descriptive characteristics of the functionality vari-ables and the TEM score variables are presented in Tables2 and 3, respectively. When the trauma intensity was in-creased, the degree of inclined plane that the rats couldclimb decreased (correlation coefficient: �0.860, p �0.001; Fig. 7 left). A similar significant negative correlationwas observed between Basso-Beattie-Bresnahan scoresand trauma intensity (correlation coefficient: �0.901, p �0.001; Fig. 7 right). The nonparametric trend test for or-dered groups showed that axonal, neuronal, and capillarydamage increased with each increasing level of trauma se-verity, as reflected by each of the five scores (p � 0.01 forall) obtained in the TEM evaluation (Fig. 8).

Discussion

Graded SCI produced using the Allen technique in therat has been confirmed by our results.2 The rats subjected



FIG. 3. Electron microscopic studies. Upper: Gray matter ob-tained in the 10-g/cm trauma group, showing near-normal nucleus(N) but fewer organelles in the cytoplasm. Lower: White matterobtained in the 10-g/cm trauma group, demonstrating greater dam-age than gray matter. The axons show some dispersion in themyelin lamella (Ad) and the mitochondria are edematous. Intactendothelium (En) and patent capillary lumen (L) are seen. Originalmagnification � 6000, bar = 1 �m.

FIG. 4. Electron microscopic studies. Upper: Gray matter ob-tained in the 25-g/cm trauma group, showing sparse chromatin andfew organelles in the nucleus. Lower: White matter obtained inthe 25-g/cm trauma group, showing edematous axons with crackedmyelin layers. Endothelial cells are swollen and lumen shows nar-rowing. Original magnification � 6000, bar = 1 �m.

to different weights dropped from the same height exhib-ited a graded functional deficit as measured by Basso-Beattie-Bresnahan scores5 and the mean angle demon-strated in the inclined-plane test of Rivlin and Tator.21

Ultrastructural results also indicated the production ofgraded lesions. These findings are well correlated with theresults presented by Wrathall, et al.,27 in which graded in-jury produced using the Allen technique was correlatedwell with clinical results and light microscopy. Basso, etal.,5 also produced graded SCI by weight-drop technique.They demonstrated that their scale can distinguish differ-ences in neurological outcomes in rats subjected to differ-ent levels of traumatic injury and that the behavioral out-come is highly correlated with histological damage.

Behavioral testing revealed changes in functional defi-cits in relation to the degree of trauma. Significant differ-ences were demonstrated among the laminectomy-only,10-g/cm trauma, and 25-g/cm trauma groups, as well asbetween the 50- and 100-g/cm groups (Table 2). Resultsobserved in the 25- and 50-g/cm trauma groups were verysimilar. Hence, we classified contusion injuries in theexperimental rat model of SCI as mild (10-g/cm), moder-ate (25–50 g/cm), and severe (100 g/cm). We found simi-lar clinical results in the laminectomy-only group pre- andpostoperatively. Because we have also previously demon-

strated that laminectomy itself has no effect on the ultra-structure of the spinal cord,15 results of nonoperated ratswere excluded.

Electron microscopy showed a gradual increase (wors-ening) in ultrastructural scores in relation to the increase oftrauma intensity. The general axonal score was the mostaffected by traumatic injury, whereas vascular endotheliumwas the least (Table 3). Although all ultrastructural compo-nents showed gradual worsening in relation to trauma in-tensity, only nucleus-related damage remained unchangedwhen injury was increased from moderate (50 g/cm) tosevere (100 g/cm). This finding is in contrast to with thatfound in the light microscopy study reported by Wrathall,et al.,27 who showed that a 50-g/cm or greater traumatic in-jury produces gray matter disappearance at the epicenter ofthe lesion 4 weeks after injury. This may be the result of theischemia-related chronic effect on gray matter. Results re-ported by Balentine, et al.,4 support our findings of earlyaxonal damage. They showed that axons in the lesionedsegment of the cord resulted in necrosis, by 8 hours postin-jury in most samples and in all samples by 1 day.

Wound healing in the central nervous system results in



53

FIG. 5. Electron microscopic studies. Upper: Gray matter ob-tained in the 50-g/cm trauma group, showing advanced nucleardamage (N) with edema (E). Lower: White matter obtained in the50-g/cm trauma group, showing severe axonal damage (A); myelinfragments (Mf), and edema (E) are seen. Original magnification �6000, bar = 1 �m.

FIG. 6. Electron microscopic studies. Upper: Gray matter ob-tained in the 100-g/cm trauma group, showing severe nuclear dam-age: empty spaces in the nucleus with loss of organelles (N), rup-tured mitochondria (M), and edema (E). Swollen endothelium (En)and completely blocked lumen (L) are also seen. Lower: Whitematter obtained in the 100-g/cm trauma group. Large area ofedema (E), complete axonal damage, erythrocytes due to red bloodcell bleeding (RBC). Original magnification � 6000, bar = 1 �m.

the formation of cysts. Guizar-Sahagun, et al.,12 havedemonstrated that there are three postinjury stages in theformation of cysts: a stage of necrosis, seen from Day 1 to1 to 2 weeks after injury; a stage of repair, from 1 to 2weeks postsurgery; and a stage of stability, from 8 to 15weeks to 1 year postlesioning. Microcystic cavities beginto develop as early as 3 days postinjury in the white andgray matter—by parenchymatous hemorrhage, vascularthrombosis, edema, axonal segmentation, and inflamma-tory infiltration. Two to 3 weeks after contusion, macro-phages, which absorb the necrotic tissue, disappear fromthe lesion site, leaving cavities of variable size. Four to 5 weeks after contusion, cysts in the trabecular system are usually well defined. Although there are some disad-vantages to measuring the cystic cavities—for example,shrinkage of cysts during histological processing or rup-ture of cysts, lack of detailed histological information, andinability to use this method for early histological exami-nation after acute SCI—the method is very useful for de-termining the pharmacological effects in chronic neuro-protection studies.6,10,14,18 The disadvantage of our presentmethod may be the use of the ultrastructural scoring sys-tem in the chronic experimental SCI. Here, if the magni-tude of the trauma is great enough to produce a cystic cav-ity within the chronic stage, it may not be possible toobtain equivalent tissue samples from the epicenter of thetraumatic injury site, as described previously. In our study,we observed no cystic cavity, probably because of theearly time period postinjury. It is also possible that mild-to-moderate injuries may not result in the formation ofhuge cysts. Hence, we propose that the present method isvery effective in the early postinjury period in cases of

mild-to-severe injuries, although it may not be as effectivein severe, chronic SCI experiments.

Labeling of cortico- and rubrospinal neurons by retro-grade tracers is a well-described and widely used methodto assess experimental SCI.9,19,20,22,26 Midha, et al.,19 havedemonstrated that counting corticospinal neurons, retro-gradely labeled with HRP, is related to the degree ofpreservation of the dorsal columns, whereas counting rednucleus neurons is related to the degree of preservation ofthe lateral columns. Naso and coworkers20 have shownthat retrogradely HRP-labeled rubrospinal neuron countsdid correlate well with functional scores in experimentalSCI. Theriault and Tator26 have proposed that red nucleusneurons exert a long-term survival effect following SCI.They recommended the use of fluorogold rather than HRPto label neurons retrogradely. Although all these methodsthat provide lesion site–related indirect information areexcellent for showing the intensity of and recovery fromthe trauma for dorsal and lateral columns, they yield nodata concerning spinal cord gray matter. They can be usedin acute as well as chronic studies, although they maypose a critical disadvantage in neuroprotection studies.Giehl and Tetzlaff11 have demonstrated that some neuro-tropic factors can prevent axotomy-induced death of cor-ticospinal neurons in the rat. Their results proved that inneuroprotection studies the pharmacological action mayaffect axotomy-induced death of cortico- or rubrospinalneurons. Therefore, with the counting of brain and mid-brain neurons in experimental drug studies, we may not beable to determine whether the drug exerts a neuroprotec-tive effect on the lesion site of the injured spinal cord orprevents the death of cerebral or midbrain neurons. In the



TABLE 3Trauma intensity scores determined using TEM

Percentiles

Score Intensity (g/cm) Means � SEMs 25 50 75

axonal myelin score 0 0.02 � 0.02 0 0 010 0.18 � 0.05 0 0 025 1.12 � 0.05 1 1 150 1.48 � 0.07 1 1 2

100 1.66 � 0.07 1 2 2general axonal score 0 0.02 � 0.02 0 0 0

10 0.58 � 0.07 0 1 125 1.48 � 0.07 1 1 250 2.26 � 0.08 2 2 3

100 2.66 � 0.07 2 3 3intracytoplasmic edema 0 0.00 � 0.00 0 0 0

10 0.20 � 0.09 0 0 025 0.55 � 0.11 0 1 150 1.35 � 0.15 1 1 2

100 1.90 � 0.16 1 2 2nucleus 0 0.00 � 0.00 0 0 0

10 0.20 � 0.09 0 0 025 0.85 � 0.17 0 1 150 1.70 � 0.15 1 2 2

100 1.70 � 0.18 1 1.5 2vascular endothelium 0 0.00 � 0.00 0 0 0

10 0.20 � 0.13 0 0 0.2525 0.40 � 0.16 0 0 150 0.80 � 0.20 0 1 1

100 1.30 � 0.15 1 1 2

* Statistically significant at p � 0.01 (nonparametric test for trends across ordered groups).

ultrastructural scoring method described in our report, it ispossible to observe the direct pharmacological action onthe lesion site by examining the quantitative and subcellu-lar data.

Spinal cord injury can also be assessed by counting ax-ons in the pyramidal tract of rats.25 The axonal countingtechnique provides quantitative information concerningthe severity of injury and is well correlated with clinicalstatus. This method has been used to evaluate acute (15-minute), and chronic (6-week) SCIs.3,9 Fehlings and Tator9

have shown that after transversely sectioning and process-ing the injury site–containing segment of spinal cord, cellcounts can be performed as long as 6 weeks after cordinjury. These data support the idea that TEM scoring ofspinal cord injury may also be possible in studies ofchronic disease.

Ultrastructural changes in neuroprotection studies havebeen widely used in acute (30-minute) and chronic (10-week) studies. Electron microscopy examination providesvery detailed information about injury site, protection of



55

FIG. 7. Graphs demonstrating the negative association observed between function and trauma intensity. Left: Thedegree of inclined plane decreases with increasing trauma intensity (correlation coefficient: �0.860, p � 0.001). Right:The Basso-Beattie-Bresnahan (BBB) scores show similar significant negative correlation with increasing trauma inten-sity (correlation coefficient: �0.901, p � 0.001).

FIG. 8. Graph showing the change in the mean TEM scores according to intensity of the traumatic injury. The scoresof axonal myelin, nucleus, vascular endothelium, axon, and edema are increased with each increasing level of traumaintensity (nonparametric trend test, p � 0.01 for all). Higher values are associated with increasing severity of SCI.

neurons, cell membranes, axons, subcellular elements,and edema.1,4,7,13,15,16 Although most of these studies lackquantitative data, it seems to be possible to evaluate theSCI site after 10 weeks.13 These data also support the useof ultrastructural scoring of SCI in chronic studies.

We previously determined that TEM scoring of SCI is auseful method in pharmacological studies.15,16 We not onlyfound that ultrastructural evaluation yielded ultrastructur-al information about the lesion site but that it allowed fornumeric comparison of control and trauma groups. It wasalso possible to compare the effects of drug actions on dif-ferent subcellular organelles.16 In those studies, we did nothave clinical data. In the present study, we demonstratedthat, when the trauma intensity was increased, behavioralresponsiveness showed a decreasing trend. A similar neg-ative correlation was observed between trauma intensityand ultrastructural scoring.

In the present study, we aimed to classify trauma inten-sity in rats in which the weight-drop technique was usedto produce SCI. Mild injury was produced using 10-g/cmtrauma, moderate injury by using 25 to 50–g/cm trauma,and severe injury by using 100-g/cm trauma according toclinical findings and quantitative ultrastructural scoring.We showed that there is a correlation between traumaintensity and quantitative ultrastructural scoring. We pro-pose that this method is useful when studying differenttrauma intensities. It can be used in experimental SCI inthe early postinjury period to evaluate detailed subcellularchanges; by using the scoring system it is possible to com-pare the effects of neuroprotective agents.

References

1. Akpek EA, Bulutcu E, Alanay A, et al: A study of adenosinetreatment in experimental acute spinal cord injury. Effect onarachidonic acid metabolites. Spine 24:128–132, 1999

2. Allen AR: Surgery of experimental lesion of spinal cord equiv-alent to crush injury of fracture dislocation of spinal column. Apreliminary report. JAMA 57:878–880, 1911

3. Anthes DL, Theriault E, Tator CH: Characterization of axonalultrastructural pathology following experimental spinal cordcompression injury. Brain Res 702:1–16, 1995

4. Balentine JD, Paris DU: Pathology of experimental spinal cordtrauma, II. Ultrastructure of axons and myelin. Lab Invest 39:254–266, 1978

5. Basso DM, Beattie MS, Bresnahan JC: Graded histological and locomotor outcomes after spinal cord contusion using theNYU weight-drop device versus transection. Exp Neurol 139:244–256, 1996

6. Braughler JM, Hall ED, Means ED, et al: Evaluation of an in-tensive methylprednisolone sodium succinate dosing regimen inexperimental spinal cord injury. J Neurosurg 67:102–105, 1987

7. Coskun K, Attar A, Tuna H, et al: Early protective effects of ilo-prost after experimental spinal cord ischemia in rabbits. ActaNeurochir (Wien) 142:1143–1150, 2000

8. Cuzick J: A Wilcoxon-type test for trend. Stat Med 4:87–90,1985

9. Fehlings MG, Tator CH: The relationships among the severity ofspinal cord injury, residual neurological function, axon counts,and counts of retrogradely labeled neurons after experimentalspinal cord injury. Exp Neurol 132:220–228, 1995

10. Fujimoto T, Nakamura T, Ikeda T, et al: Effects of EPC-K1 onlipid peroxidation in experimental spinal cord injury. Spine 25:24–29, 2000

11. Giehl KM, Tetzlaff W: BDNF and NT-3, but not NGF, preventaxotomy-induced death of rat corticospinal neurons in vivo.Eur J Neurosci 8:1167–1175, 1996

12. Guizar-Sahagun G, Grijalva I, Madrazo I, et al: Development ofpost-traumatic cysts in the spinal cord of rats subjected to se-vere spinal cord contusion. Surg Neurol 41:241–249, 1994

13. Ildan F, Polat S, Oner A, et al: Effects of naloxone on sodium-and potassium-activated and magnesium-dependent adenosine5�-triphosphate activity and lipid peroxidation and early ultra-structural findings after experimental spinal cord injury. Neu-rosurgery 36:797–805, 1995

14. Kao CC, Chang LW, Bloodworth JM: The mechanism of spinalcord cavitation following spinal cord transection. Part 2. Elec-tron microscopic observations. J Neurosurg 46:745–756, 1977

15. Kaptanoglu E, Caner HH, Surucu SH, et al: Effect of mexiletineon lipid peroxidation and early ultrastructural findings in experi-mental spinal cord injury. J Neurosurg (Spine 2) 91:200–204,1999

16. Kaptanoglu E, Tuncel M, Palaoglu S, et al: Comparison of theeffects of melatonin and methylprednisolone in experimentalspinal cord injury. J Neurosurg (Spine 1) 93:77–84, 2000

17. Kirkali Z, Esen AA, Hayran M, et al: The effect of extracorpo-real electromagnetic shock waves on the morphology and con-tractility of rabbit ureter. J Urol 154:1939–1943, 1995

18. Means ED, Anderson DK, Waters T, et al: Effect of methyl-prednisolone in compression trauma to the feline spinal cord. JNeurosurg 55:200–208, 1981

19. Midha R, Fehlings MG, Tator CH, et al: Assessment of spinalcord injury by counting corticospinal and rubrospinal neurons.Brain Res 410:299–308, 1987

20. Naso WB, Cox RD, McBride JP, et al: Rubrospinal neurons andretrograde transport of fluoro-gold in acute spinal cord injury—a dose-response curve. Neurosci Lett 155:125–127, 1993

21. Rivlin AS, Tator CH: Effect of duration of acute spinal cordcompression in a new acute cord injury model in the rat. SurgNeurol 10:38–43, 1978

22. Ross IB, Tator CH, Theriault E: Effect of nimodipine or methyl-prednisolone on recovery from acute experimental spinal cordinjury in rats. Surg Neurol 40:461–470, 1993

23. Schwab ME, Bartholdi D: Degeneration and regeneration of ax-ons in the lesioned spinal cord. Physiol Rev 76:319–370, 1996

24. Tasdemir O, Katircioglu F, Kucukaksu S, et al: Warm bloodcardioplegia; ultrastructural and hemodynamic study. AnnThorac Surg 56:305–311, 1993

25. Tator CH, Rivlin AS, Lewis AJ, et al: Effect of acute spinal cordinjury on axonal counts in the pyramidal tract of rats. J Neu-rosurg 61:118–123, 1984

26. Theriault E, Tator CH: Persistence of rubrospinal projectionsfollowing spinal cord injury in the rat. J Comp Neurol 342:249–258, 1994

27. Wrathall JR, Pettegrew RK, Harvey F: Spinal cord contusion inthe rat: production of graded, reproducible, injury groups. ExpNeurol 88:108–122, 1985

Manuscript received August 27, 2001.Accepted in final form February 26, 2002.Address reprint requests to: Selcuk Palaoglu, M.D., Department

of Neurosurgery, Hacettepe University, School of Medicine, 06100-Sihhiye, Ankara, Turkey. email: [email protected].



ultrastructural scoring of graded acute spinal cord injury in the rat

Documents