Three-dimensional morphometry of spinal cord injury following polyethylene glycol treatment
Center for Paralysis Research, Institute for Applied Neurology, Department of Basic Medical Sciences, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907-1244, USA
*e-mail: cpr{at}vet.purdue.edu
Accepted 16 October 2001
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Summary |
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Key words: three-dimensional reconstruction, computer visualization, morphometry, spinal cord injury, neurotrauma, cavitation.
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Introduction |
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It is believed that more extensive damage to the spinal cord occurs with time after the initial trauma by a progressive and delayed secondary injury process (Blight, 1992, 1993
; Honmou and Young, 1995
). Since the application of PEG rescues significant behavioral and physiological function in every animal treated, we believed this would be associated with a considerable rescue of spinal cord parenchyma through the sealing of damaged cell membranes. This idea was tested in the present study using histological data sets obtained from animals that were part of the study of the delayed application of PEG (Borgens et al., 2002
) as well as an equivalent number of animals obtained from studies of the immediate application of PEG following injury (Borgens and Shi, 2000
).
The injury site of the chronically traumatized mammalian spinal cord is located predominantly in the central regions of the spinal cord, making three-dimensional anatomical description of the lesion difficult. Previous histological techniques have required the investigator to infer three-dimensional morphology from a series of two-dimensional sections. In addition, measuring these three-dimensional structures from serial sections has required the use of two-dimensional morphometry or other approximation methods (Blight, 1985; Noble and Wrathall, 1985
; Harris and Stevens, 1988
; Hashimoto and Kimura, 1988
; Bresnahan et al., 1991
; Halliday et al., 1993
; Arndt et al., 1994
; Navarro et al., 1994
; Salisbury, 1994
). Here, we applied novel three-dimensional algorithms to generate computer models of the injured spinal cord developed from complete sets of the serial histological sections comprising a spinal cord segment containing the lesion. This software allowed the visualization of the three-dimensional shape and pathological structures of injured cords and, to our knowledge, is the only means of measuring the surface area and volume of pathological features of interest from the reconstruction itself (Duerstock et al., 2000
). These new computer reconstruction techniques have already proved to be ideal for morphometrical evaluation of complex histological material (Moriarty et al., 1998
).
Since 100 % of the control (untreated) population failed to recover conduction through the compression lesion, while 100 % of the PEG-treated animals recovered conduction swiftly, and since 93 % of PEG-treated animals recovered behavioral function compared with infrequent spontaneous recovery in controls, it was likely that every PEG-treated spinal cord would be markedly different from sham-treated spinal cords in other aspects of evaluation. Thus, data sets from four animals from an immediate PEG-treated group and six animals from a delayed PEG-treated group were reconstructed and compared with a set of four control animals. We found that PEG application statistically significantly increased the amount of spared parenchyma in injured spinal cords while also significantly reducing the degree of cavitation and cyst formation.
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Materials and methods |
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Animals
Adult (300 g) laboratory guinea pigs were used in these experiments. Following surgery (see below), they were housed two animals per cage and fed ad libitum; their health was monitored daily. Seventeen animals in this study were killed 1 month post-treatment by anesthetizing them with 0.2 ml of ketamine HCl and 0.2 ml of xylazine, then by an overdose of sodium pentobarbital (0.8 ml of 1 g ml1 standard injectable) immediately followed by perfusion/fixation with 6 % paraformaldehyde, 0.5 % glutaraldehyde in phosphate buffer. Segments of spinal cord containing the injury sites were dissected free and immersion-fixed in the above fixative for approximately 18 h.
Surgical procedures
Anesthesia was performed using an intramuscular injection of 0.1 ml 100 g1 body mass of a standardized solution of 100 mg kg1 of ketamine HCl and 20 mg kg1 of xylazine. The spinal cord was exposed by a partial laminectomy (between the tenth and twelfth thoracic vertebrae), the dura was removed with sharpened watchmakers forceps and the dorsal hemisphere was compressed for 15 s using blunted watchmakers forceps possessing a détente to standardize the displacement of the spinal cord (Blight, 1991; Moriarty et al., 1998
). For five spinal cords in one experimental group, PEG (Mr 1800, 50 % w/v in water) was applied to the injury site with a pipette immediately after the crush. The injury site was rinsed after 2 min with Krebs solution to remove the PEG. In a second experimental group consisting of seven spinal cords, PEG was applied approximately 7 h post-injury before being rinsed off. In sham-treated control animals, water (vehicle) was immediately applied to the lesion for 2 min and aspirated. The injury site was then lavaged with Krebs solution. Surgical incisions were closed in layers with 3-0 proline suture, and the skin was closed with wound clips. Immediately post-surgery, each animal was injected subcutaneously with 3 ml of lactated Ringers solution to prevent dehydration, and the animal was placed under a heat lamp for approximately 24 h to reduce post-surgical mortality due to shock.
Histological preparation
The excised segments of spinal cord (approximately one vertebral segment; approximately 1 cm in length) including the experimental injury were dehydrated in ascending concentrations of alcohol followed by xylene to allow infiltration and embedding in Paraplast (paraffin) by conventional methods. This piece of cord was reduced in length by removing undamaged tissue at the rostral and caudal ends, yielding a segment length of approximately 4.2 mm containing the lesion in its center. This shorter segment facilitated histological sectioning and the retrieval of these serial sections. Even minor imperfections in the retrieved sections (that would be of little consequence to two-dimensional evaluation and morphometry) can spoil the precision of the three-dimensional registration and reconstruction process.
This entire cord segment was then sectioned on a rotary microtome at approximately 15 µm per section, and horizontal longitudinal sections were affixed to microscope slides. Prior to use, the slides were dipped in a 0.5 % (w/v) gelatine solution that aids the adhesion of the sections to the slides during subsequent treatment. Paraffin was partially removed with a 1 h treatment in an oven at 60°C and completely removed after a 1 h immersion in 100 % xylene. Sections were rehydrated by immersions in descending concentrations of alcohol in distilled water using conventional methods. All sections were stained with Holmes silver stain, counterstained with Neutral Red and coverslipped in Permount. The silver stain clearly revealed intact white and gray matter, while the counterstain delineated the region of central hemmorhagic necrosis. Cysts were revealed by the absence of stain.
Video capturing and registration of serial sections
An Optronics DEI-750 color video camera mounted on an Olympus Van Ox Universal microscope displayed histological sections on a computer monitor. Histological images were then acquired to a dual Pentium Pro computer using Adobe Photoshop software and managed on a Silicon Graphics Indigo for three-dimensional surface reconstruction. Registration was performed by superimposing each successive histological section by optimally positioning and rotating the microscope stage onto a tracing of the previously captured image made on the computer monitor. The boundaries of the spinal cord, cysts and lesion site served as effective fiducial markers.
Three-dimensional reconstruction
As mentioned above, all retrieved histological sections obtained from these spinal segments were used during three-dimensional reconstruction. Only a few of the most dorsal and ventral sections were lost as the microtome advanced into and out of the tissue. This minor loss of sections made the reconstructed images appear flattened on their most dorsal and ventral surfaces (see below). Serial histological sections must be in excellent condition to permit effective registration and/or reconstruction. For this reason, one spinal cord data set was chosen, and deleted, from each of the three groups (control, immediate PEG application and delayed PEG application) that was not of the quality of the remaining sets. This gave a final set of four control, four immediate PEG application and six delayed PEG application spinal cord.
The isocontouring algorithm proposed by Bajaj et al. (1997) was used for all three-dimensional reconstruction. We chose this novel three-dimensional reconstruction algorithm rather than commercially available software to image the spinal cord injuries three-dimensionally because of its ease of use and ability to interrogate reconstructed samples and objects embedded within the samples quantitatively (Duerstock et al., 2000
). For, reference, Fig. 1 demonstrates the ability of the isocontouring method to produce an effective three-dimensional image of an undamaged spinal cord segment from serial histological sections.
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All computer capture of data sets was performed on sections given an identification code by a technician. The identity of all spinal cord data sets (control, immediate PEG application or delayed PEG application) was unknown to the investigators. One investigator (BD) was also blind to the identity of experimental and control spinal cord injuries during isovalue selection, as mentioned above, and subsequent production and quantitative interrogation of the three-dimensional image. The identity of none of the images was revealed until all three-dimensional visual images had been finalized, quantitatively queried and the numerical data organized in spreadsheets.
Three-dimensional morphometric analysis
We measured regions of intact spinal cord parenchyma, lesion formation and cavitation on the basis of differences in their pixel density in both PEG-treated and sham-treated spinal cords. Intact white matter appeared dark red to reddish/black at low magnification because of the black staining of axons set against a Neutral Red counterstain. The lesion could be identified by its pale staining since it was devoid of Holmes-silver-impregnated neurons and axons (Fig. 2).
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Statistical evaluation
The acceptable use of a small sample size from each comparator (treatment) group was described above. We further substantiated this number of subjects by using a one-tailed, Students t-distribution equation:
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where S is standard deviation, =ß=0.1 and
(the success criterion) is 30 % or greater. Fewer than four subjects are required in each comparator group, which is below the sample size we used, to reach statistical significance.
The normalized measurements of the three-dimensional reconstructions between control and experimental groups were compared using an unpaired, two-tailed, Students t-test. Pearson correlation tests were used to test linear relationships. Computations were performed using Instat software.
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Results |
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The three-dimensional reconstruction in Fig. 4 shows both the three-dimensional image and two-dimensional photomicrographs typical of regions of (i) destroyed central gray matter, (ii) marginal areas of spared gray and white matter and (iii) the subpial region of spared white matter. An evaluation of these images relative to the three-dimensional reconstructions provides the basis for the comparisons noted below. Surviving subpial regions were almost exclusively white matter; however, a thorough search revealed rare examples of spared neurons (Fig. 4B) in regions of adjacent white matter and gray matter.
In PEG-treated spinal segments, normal-looking parenchyma (both gray and white) was more abundant than in untreated spinal cords (Figs 4, 5). The mean volume of normal-looking parenchyma after trauma in untreated spinal segments was 24.4 %, while 35.8 % of the nervous tissue survived after treatment in the immediate spinal cords and 49.7 % in delayed PEG-treated spinal cords (see Fig. 7A).
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Using the normalized volume measurements derived from the control and PEG-treated groups, we performed correlation tests to determine whether there was a relationship between the sparing of parenchyma, the occurrence of cysts and the formation of the lesion. These tests resulted in high correlation coefficients between the amount of intact tissue and the size of the lesion in untreated (r=0.81), immediate PEG-treated (r=0.94) and delayed PEG-treated (r=1.00) spinal cord segments (Fig. 8A). However, there did not appear to be a linear correlation between the volume of intact parenchyma and total cyst volume for untreated (r=0.19), and immediate PEG-treated (r=0.57) groups (Fig. 8B). Correlation tests were also performed between cavitation and lesion size for control (r=0.42), immediate PEG-treated (r=0.81) and delayed PEG-treated (r=0.86) groups (Fig. 8C). Only the correlation coefficient for the delayed PEG-treated group was statistically significant (P0.05).
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Discussion |
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Three-dimensional methodology
We believe that the use of every histological section comprising the data set for each spinal cord during three-dimensional reconstruction, the absence of human interaction in the gathering of data and the precise quantitative querying of the three-dimensional model produced reliable visualizations and numerical data for comparison. The anatomical structures of the injured spinal cord segments that we imaged and quantified were delineated on pixel value differences in the histological image. This eliminated the subjective method of identification of anatomy as well as the widespread manual tracing of structures of interest by an investigator prior to three-dimensional reconstruction and quantification. These differences in pixel values were based on the very different staining characteristics of intact and destroyed gray and white matter and the absence of staining that revealed cysts in relief. This approach is less biased and more precise than other three-dimensional reconstruction methods applied to spinal cord injury that require manual tracing of regions of interest for each section and the morphing of regions of spinal tissue between the relatively few histological sections used to produce the three-dimensional model. In many such studies, only one histological section in ten is registered and imaged, producing a visualization that is questionably 90 % computer-extrapolated (Halliday et al., 1993; Martone et al., 1993
; Navarro et al., 1994
; Liss, 1995
; Beattie et al., 1997
). We have reviewed and compared several three-dimensional reconstruction methods, including the algorithm used here, with others in the literature (Moriarty et al., 1998
; Duerstock et al., 2000
).
The three-dimensionally reconstructed injury
Because of the severity of the compression injury, the overall damage to the spinal cords was very pronounced. Cavitation in most animals was especially prevalent. We found that cavitation in the untreated spinal cords was significantly greater than in the PEG-treated groups. In the control spinal cords, cysts usually extended throughout most of the cross-sectional area of the spinal cord segment and beyond. Thus, the quantification of cysts was very conservative, underestimating their size, which subsequent to correction would have increased the statistical difference between controls and experimentals.
The total number of histological sections per data set varied among the spinal cord segments that we reconstructed three-dimensionally. The difference in the number of sections between control and delayed PEG-treated spinal cords resulted from their different pathologies rather than from differences in histological preparation. In all three-dimensional reconstructions, the entirety of the injury site was contained within the data set of histological sections. The control cords were more cavitated than the experimental cords and, in some cases, cysts extended out of the section in the rostral/caudal direction. Curiously, it appears that cysts generally helped maintain the roughly cylindrical shape of the spinal cord better than in less cavitated PEG-treated cords. This may have been because cysts increased the turgidity of the spinal segment by a ballooning effect, producing a larger-diameter segment to be histologically sectioned (Figs 24). In contrast, the PEG-treated spinal cord segments tended to form a compact hourglass shape requiring less sectioning to produce a complete data set (see Figs 25). Indeed, although the PEG-treated spinal segments were smaller in diameter, they contained a greater absolute volume of both intact and damaged nerve tissue than the control spinal cord segments.
Intact regions of both white and gray matter were also more prevalent in PEG-treated cords. We emphasize that, while spared parenchyma was not exclusively white matter (see Fig. 4B), examples of surviving neurons in gray matter was rare. This is due to the centrifugal spread of tissue loss during central hemorrhagic necrosis, since gray matter is richly vascularized (Allen, 1911), and the centralized focus of force when a cylinder of a sol/gel (i.e. spinal cord) is impacted from the outside. Nonetheless, the overall lesion was smaller in PEG-treated cords than in controls. This suggests that PEG may also seal damaged neuronal (and other cell body) membranes in addition to their processes. This has been directly tested in vitro using a related polymer, Poloxamer 188. Application of this triblock (polyethylenepolypropylenepolyethylene) polymer provided potent neuroprotection to hippocampal and cerebellar neurons in culture exposed to oxidative and excitoxic injury (Marks et al., 2001
). PEG-mediated sparing in dorsal regions of white matter probably contributed to the striking recovery of somatosensory evoked potential conduction following tibial nerve stimulation relative to a complete lack of such conduction in control animals (Borgens et al., 2002
; Borgens and Shi, 2000
).
The lesion itself was another structure of interest. Damaged or destroyed spinal cord parenchyma was largely devoid of silver-stained neurons or their processes, appearing pale red in comparison. In PEG-treated spinal cords, the lesion was localized in the center of the site of injury. In untreated spinal cords, the lesion was less localized and was spread diffusely throughout the entire spinal segment surrounding the more numerous cysts. This observation was supported by measurements of the extended surface area of the lesion in control cords relative to treated cords.
Three-dimensional quantification
There was a statistically significant decrease in the surface area of the lesion in PEG-treated spinal segments compared with untreated spinal segments, as discussed above. The volume of intact spinal cord parenchyma was found to be significantly greater in PEG-treated animals than in untreated animals, while the volume of cavitation was significantly smaller. The application of PEG to compressed spinal cords may have resulted in the rescue of compromised cells, leading to a reduction in the spread of cavitation.
We investigated whether the lesion and cysts in PEG-treated and untreated spinal cords were related to the amount of spared parenchyma. It appeared that an increased amount of intact spinal cord tended to be correlated to a decrease in the size of the lesion in experimental and control groups, as one might expect. This was definitely true for delayed PEG-treated animals, for which regression analysis resulted in markedly statistically significant correlation coefficients (P=0.0001; see Fig. 8A). However, no direct relationship was evident between the volume of intact parenchyma and the volume of cysts that formed within injured spinal cords, except in the delayed PEG-treated group (Fig. 8B).
We have not shown conclusively that, with increasing cavitation, lesion formation decreased, although a trend in this direction occurred in the PEG-treated groups (Fig. 8C). For the delayed PEG-treated animals, the correlation test proved statistically significant (P=0.03). In control spinal cords, the process of cavitation did not appear to be directly related to the volume of the lesion. Although cell death and the resultant inflammatory reaction may initiate cavitation, there are certainly other factors that control its extent (see Perry et al., 1987, 1993
; Leskovar et al., 2000
).
Finally, these data provide insight into the gross anatomical effects of a brief PEG treatment to the compression-injured spinal cord. In addition to (i) a very rapid and measurable recovery of physiological and behavioral function (Borgens and Shi, 2000; Borgens et al., 2002
), (ii) a rapid sealing of membrane breaches to individual axons within white matter revealed by a dye-exclusion test (Shi and Borgens, 2001
), we can add (iii) that the total amount of intact parenchyma is augmented and (iv) that the extent of cystic cavitation is significantly reduced by PEG treatment.
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Acknowledgments |
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References |
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