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DOI: 10.1148/radiol.2421050680
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(Radiology 2007;242:129-136.)
© RSNA, 2007

Experimental Studies

Early Ischemia in Growing Piglet Skeleton: MR Diffusion and Perfusion Imaging1

Nina M. Menezes, PhD, Susan A. Connolly, MD, Frederic Shapiro, MD, Elizabeth A. Olear, MA, Rafael M. Jimenez, MD, David Zurakowski, PhD and Diego Jaramillo, MD, MPH2

1 From the Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, 149 13th St, Rm 2301, Boston, MA 02129 (N.M.M.); Departments of Radiology (S.A.C.), Orthopaedic Surgery (F.S., D.Z.), and Pathology (F.S.), Children's Hospital Boston and Harvard Medical School, Boston, Mass; and Division of Pediatric Radiology, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, Mass (E.A.O., R.M.J., D.J.). Received April 24, 2005; revision requested June 21; revision received October 30; accepted December 15; final version accepted March 20, 2006. Supported by NIH grant R01 AR042396-09. Address correspondence to N.M.M. (e-mail: nmenezes{at}nmr.mgh.harvard.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Purpose: To determine whether diffusion changes with ischemia of increasing duration, whether diffusion magnetic resonance (MR) imaging provides different information than does gadolinium-enhanced imaging, and which structural and/or biochemical changes are potentially responsible for any changes in diffusion.

Materials and Methods: Ischemia was surgically induced in one hip of each piglet (n = 8) after approval from the Subcommittee on Research Animal Care; the other hip served as a control. Piglets were imaged at approximately 48 hours and 1, 2, 4, and 8 weeks after surgery at 1.5 T by using line-scan diffusion and dynamic gadolinium-enhanced MR imaging. Apparent diffusion coefficients (ADCs) and enhancement ratios (ERs) were calculated. Significant differences in ADC and ER values over time were evaluated by using the Student t test (P < .05). At 8 weeks, piglets were sacrificed for histologic evaluation.

Results: MR images of ischemic hips showed essentially no flow 48 hours after surgery. Spontaneous partial reperfusion was observed 1–4 weeks after surgery (ischemic ER/control ER = 66% ± 35 [standard deviation]), and the ER of the ischemic hips was well above that of the control hips at 8 weeks. The ADC of ischemic hips was elevated above that of control hips before reperfusion 1 week after surgery by 47% ± 12 and remained elevated despite flow restoration. Gross structural abnormalities on MR images appeared to coincide with reperfusion. Histologic findings revealed abnormal epiphyseal cartilage thickening, cartilaginous islands within ossified tissue, and less fatty marrow in ischemic hips than in control hips; all of these factors could explain elevated ADC.

Conclusion: Diffusion is sensitive to early ischemia and follows a different time course than that of changes observed with gadolinium enhancement. ADC remained elevated in this model of severe, prolonged ischemia despite the spontaneous partial restoration of blood flow seen on gadolinium-enhanced images.

© RSNA, 2007


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Ischemia of the growing ends of long bones that leads to avascular necrosis is a common pathway for a number of disorders that result in deformity and disability in childhood and predispose to osteoarthritis in later life. Ischemia of sufficient severity and duration in the proximal femur (hip) disrupts the integrity of the epiphysis and physis and results in abnormal growth, structural deformity, and, ultimately, joint destruction. In children, femoral head ischemia occurs frequently. It can arise for unknown reasons as in Legg-Calvé-Perthes disease, during the course of diseases affecting marrow vascularity, or as a result of treatment for developmental hip dysplasia and slipped capital femoral epiphysis. The long-term morbidity of ischemic episodes can be substantial and often leads to multiple childhood surgeries and total hip arthroplasty in adult years. Skeletal ischemia is diagnosed primarily by observing evidence of bone destruction and collapse at radiography, abnormal marrow signal at magnetic resonance (MR) imaging, or abnormal uptake at scintigraphy. More recently, gadolinium-enhanced MR imaging has been used to demonstrate lack of adequate blood flow (1–4). Lack of blood flow alone, however, does not indicate the severity of damage or provide a prognosis as to the fate of the tissue. Some cases of skeletal ischemia resolve spontaneously, whereas others require intervention to restore flow and/or minimize long-term disability. Furthermore, reperfusion across the growth-plate cartilage results in premature growth-plate closure and worse outcome (5).

In epiphyseal and growth-plate cartilage, water is organized around macromolecules and tissue disruption would likely affect water diffusion. Diffusion-weighted MR imaging has been shown to be sensitive at depicting brain ischemia, particularly tissue destined for infarction in acute stroke (6,7). Diffusion-weighted MR imaging reflects the presence and duration of short-term skeletal ischemia in an animal model (8). We hypothesized that diffusion-weighted MR imaging findings could serve as a prognostic marker of skeletal ischemia prior to the manifestation of gross anatomic changes and independent of the status of epiphyseal blood flow (5). Thus, the purpose of our study was to determine whether diffusion changes with ischemia of increasing duration, whether diffusion MR imaging provides different information than does gadolinium-enhanced imaging, and which structural and/or biochemical changes are potentially responsible for any changes in diffusion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Animal Model
Our study was conducted with approval from the Massachusetts General Hospital Subcommittee on Research Animal Care and in compliance with National Institutes of Health guidelines. Ischemia was induced in the right hip of eight piglets by surgically disrupting the arterial supply of the femoral epiphysis with ligation of the femoral neck vessels and resection of the ligamentum teres femoris (F.S.). This procedure results in histologic changes that closely resemble those of Legg-Calvé-Perthes disease (8,9–12). The nonischemic (left) hip was the internal control. The piglets were 3 weeks old at the start of the study so the skeletal maturity would correspond to that of a human child 3–5 years old.

Prior to MR imaging, the piglets were anesthetized with an intramuscular injection of 40 mg of midazolam hydrochloride (Baxter, Deerfield, Ill) and 20 mg of ketamine hydrochloride (Abbott Laboratories, North Chicago, Ill) per kilogram of body weight. A second dose of anesthetic that comprised 20 mg/kg ketamine hydrochloride and 20 mg/kg xylazine (Xyla-ject; Phoenix, St. Joseph, Mo) was administered approximately 30 minutes later. This was followed by continuous intravenous infusion of propofol 1% (Diprivan; Baxter, Irvine, Calif) diluted in normal saline at 0.002 (mg · kg–1)/min. The animals were imaged with an oral airway in place and received blow-by oxygen at 4 L/min.

MR Imaging
MR imaging was performed at 1.5 T (GE Medical Systems, Milwaukee, Wis) by using a pair of 3-inch receive-only surface coils at approximately 48 hours and 1, 2, 4, and 8 weeks after surgery. In all animals, conventional T1-weighted, T2-weighted, and spoiled gradient-recalled echo images were obtained with 2.5-mm section thickness, 0.625-mm in-plane resolution, and 20-cm field of view. T1-weighted images were acquired with repetition time msec/echo time msec of 500/9 and one signal acquired. T2-weighted images were acquired with 2000/60 and two signals acquired.

Diffusion-weighted MR imaging was performed by using line-scan diffusion imaging (13–15). With this technique, multiple diffusion-weighted spin-echo column excitations were used to construct a two-dimensional image. Line-scan diffusion imaging was used to measure diffusion in six directions by using 2100/70, b = 0 and 700 mm2/sec, four signals acquired, 4-mm section thickness, 1.25-mm in-plane resolution, and 20-cm field of view.

Gadopentetate dimeglumine (Magnevist; Berlex, Wayne, NJ) at 0.2 mmol/kg was injected manually in a rapid bolus into an ear vein 10 seconds after the beginning of dynamic gadolinium-enhanced MR imaging. The injection lasted approximately 5 seconds; injected volumes ranged from 2.8 to 3.2 mL. Enhancement was evaluated by using a spoiled gradient-echo MR sequence (200/2; flip angle, 60°; section thickness, 3 mm; in-plane resolution, 0.625 mm; field of view, 20 cm). Five images were acquired per section and 78 seconds apart. Postcontrast spin-echo T1-weighted images (500/9) also were obtained.

Image Analysis
From the diffusion-weighted MR images, the apparent diffusion coefficient (ADC) was calculated (7). Enhancement ratios (ERs) comparing the signal intensity (SI) before and after the administration of intravenous contrast material were calculated as ER = (SIpeak – SIbaseline)/SIbaseline, where SIpeak is the maximum SI after injection of contrast material and SIbaseline is the precontrast SI.

Because of the marked, progressive anatomic deformity that occurred over time in this animal model, separate analyses for the different compartments of the femoral head (epiphyseal cartilage, secondary center of ossification, and physis) proved to be difficult at late time points. Therefore, the ER and ADC values provided for each animal at each time point are average values for a region of interest (N.M.M.) that comprises all the compartments of the femoral head.

We evaluated the SI changes on T1- and T2-weighted images. One observer (S.A.C.) compared the SI of the ischemic femoral head with that of the control femoral head. SI was recorded as unchanged, increased, or decreased when compared with that of the control hip.

Histologic and Anatomic Measurements
At the end of the 8-week study, the piglets were sacrificed and femurs were removed for gross and histologic evaluation (F.S., 25 years of experience in bone pathology). The animals were sacrificed with an intracardiac injection of pentobarbital sodium (Nembutal; Abbott Laboratories) at 2 mg/kg immediately after the last imaging session. The proximal femurs were cut at the metaphyseal level, freed from attached soft tissues, and fixed in 10% neutral buffered formalin for 2 weeks. The bones then were decalcified in 25% formic acid until soft. The bone ends were cut with a razor in the midcoronal plane, photographed, and cut into smaller pieces to assess specific regions for structural changes. Structural studies (F.S.) of ischemic and control femurs involved gross photographs, specimen radiographs, photographs of coronal plane hemisections after decalcification, and photomicrographs of 5-µm-thick slices after preparation with paraffin embedding and plastic (JB4; Polysciences, Warrington, Pa) embedding techniques (3).

Prior to cutting the femurs, femoral head and neck shortening were determined with measurements from the proximal femoral head articular surface to the distal femoral articular surface in exposed ischemic and control femurs.

Statistical Analysis
All continuous variables were found to follow a Gaussian distribution by using the Kolmogorov-Smirnov statistic (16) and therefore are expressed as mean ± standard deviation. Significant differences in ER and ADC values were assessed by using paired t tests because the ischemic and control hips were within the same animal (17). Repeated-measures analysis of variance by using the Greenhouse-Geisser F test for small samples was applied to determine changes in ER and ADC values over time, which takes into account the longitudinal nature of the data and the within-animal correlation (18). To handle the issue of multiple testing, the conservative two-tailed P < .01 was considered to indicate a statistically significant difference to protect against type I (false-positive) errors due to multiple time point comparisons (19).

Statistical analysis was performed with software (SPSS, version 13.0; SPSS, Chicago, Ill). Results of power analysis indicated that a sample size of eight animals would provide 80% power (ß = 0.20) to detect a 25% difference in ER and ADC values between ischemic hips and control hips with the assumption of a standard deviation of 15% (effect size, 1.67); a paired t test with a two-tailed significance level of .01 was used (nQuery Advisor, version 6.0; Statistical Solutions, Cork, Ireland).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Control Hip Values
Enhancement on images of the control hips (n = 8) was high at 48 hours after surgery; ER was 1.20 ± 0.31 for the entire femoral head (epiphyseal cartilage, secondary center of ossification, and physis). Images of the control hips showed some variation in ER values during the study, although there were no systematic changes. ADC values of the control hips at 48 hours after surgery were consistent with those of prior studies (8); ADC was 1.05 ± 0.25. ADC values of the control hips declined significantly over time (analysis of variance F = 17.92, P < .001). On T1-weighted images of the control hips, SI at 3 weeks of age was between that of muscle and that of subcutaneous fat and similar to that of the metaphysis; SI gradually increased over time. SI changes during the study were not observable on T2-weighted images.

Ischemic Hip Values
In all animals, femoral neck ligature led to markedly decreased blood flow in the ischemic hips at 48 hours after surgery (Figs 1, 2). Results of paired t tests revealed large significant differences between ERs of ischemic hips and ERs of control hips at 48 hours (P < .001), 1 week (P < .001), and 2 weeks (P < .01) after surgery (Table, Fig 3a). Spontaneous partial reperfusion of the ischemic femoral head was first observed (as increased SI on postcontrast T1-weighted images) at 1–4 weeks after surgery and continued throughout the study. No significant differences between ERs of ischemic hips and those of control hips were observed at 4 weeks (P = .90) and 8 weeks (P = .07) after surgery. This indicated that the ischemic hips had ER values comparable to those of the control hips at approximately 4 weeks and 8 weeks after surgery (Table). Reperfusion occurred in a spatially heterogeneous manner (Fig 2) that varied from time point to time point, as well as from animal to animal. Reperfused regions tended to have higher ER values than the control hips; this suggests overcompensation. At the end of the 8-week study, ER values of the ischemic hips were higher than those of the control hips, although this difference was not statistically significant.


Figure 1
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  		Figure 1a: Top row: Coronal T1-weighted spin-echo (500/9) MR images obtained after administration of gadolinium-based contrast material. Bottom row: Line-scan diffusion MR imaging (2100/70, b = 0 and 700 mm2/sec) ADC maps. (a) Images of control hip 1 week after surgery show normal enhancement. (b) Images of ischemic hip at 1 week show complete loss of blood flow to femoral head and increased diffusion (arrow). (c) Images of control hip at 8 weeks. (d) Images of ischemic hip at 8 weeks. Gross morphologic changes (smaller, broadened head) are evident in ischemic hip. Portions of ischemic femoral head appear bright and indicate revascularization (dark region to right of head is fluid, which indicates increase in joint space). ADC has remained elevated in ischemic head when compared with that of control head despite restoration of blood flow.

 

Figure 1
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  		Figure 1b: Top row: Coronal T1-weighted spin-echo (500/9) MR images obtained after administration of gadolinium-based contrast material. Bottom row: Line-scan diffusion MR imaging (2100/70, b = 0 and 700 mm2/sec) ADC maps. (a) Images of control hip 1 week after surgery show normal enhancement. (b) Images of ischemic hip at 1 week show complete loss of blood flow to femoral head and increased diffusion (arrow). (c) Images of control hip at 8 weeks. (d) Images of ischemic hip at 8 weeks. Gross morphologic changes (smaller, broadened head) are evident in ischemic hip. Portions of ischemic femoral head appear bright and indicate revascularization (dark region to right of head is fluid, which indicates increase in joint space). ADC has remained elevated in ischemic head when compared with that of control head despite restoration of blood flow.

 

Figure 1
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  		Figure 1c: Top row: Coronal T1-weighted spin-echo (500/9) MR images obtained after administration of gadolinium-based contrast material. Bottom row: Line-scan diffusion MR imaging (2100/70, b = 0 and 700 mm2/sec) ADC maps. (a) Images of control hip 1 week after surgery show normal enhancement. (b) Images of ischemic hip at 1 week show complete loss of blood flow to femoral head and increased diffusion (arrow). (c) Images of control hip at 8 weeks. (d) Images of ischemic hip at 8 weeks. Gross morphologic changes (smaller, broadened head) are evident in ischemic hip. Portions of ischemic femoral head appear bright and indicate revascularization (dark region to right of head is fluid, which indicates increase in joint space). ADC has remained elevated in ischemic head when compared with that of control head despite restoration of blood flow.

 

Figure 1
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  		Figure 1d: Top row: Coronal T1-weighted spin-echo (500/9) MR images obtained after administration of gadolinium-based contrast material. Bottom row: Line-scan diffusion MR imaging (2100/70, b = 0 and 700 mm2/sec) ADC maps. (a) Images of control hip 1 week after surgery show normal enhancement. (b) Images of ischemic hip at 1 week show complete loss of blood flow to femoral head and increased diffusion (arrow). (c) Images of control hip at 8 weeks. (d) Images of ischemic hip at 8 weeks. Gross morphologic changes (smaller, broadened head) are evident in ischemic hip. Portions of ischemic femoral head appear bright and indicate revascularization (dark region to right of head is fluid, which indicates increase in joint space). ADC has remained elevated in ischemic head when compared with that of control head despite restoration of blood flow.

 

Figure 2
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  		Figure 2a: Top row: Coronal T1-weighted spin-echo (500/9) MR images obtained after administration of gadolinium-based MR contrast material. Bottom row: Line-scan diffusion MR imaging (2100/70, b = 0 and 700 mm2/sec) ADC maps. (a) Images of control hip 1 week after surgery show normal enhancement and diffusion. (b) Images of ischemic hip 1 week after surgery show complete loss of blood flow and increased ADC to femoral head. (c) Images of ischemic hip 2 weeks after surgery show partial, heterogeneous reperfusion (arrows) and heterogeneously elevated ADC.

 

Figure 2
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  		Figure 2b: Top row: Coronal T1-weighted spin-echo (500/9) MR images obtained after administration of gadolinium-based MR contrast material. Bottom row: Line-scan diffusion MR imaging (2100/70, b = 0 and 700 mm2/sec) ADC maps. (a) Images of control hip 1 week after surgery show normal enhancement and diffusion. (b) Images of ischemic hip 1 week after surgery show complete loss of blood flow and increased ADC to femoral head. (c) Images of ischemic hip 2 weeks after surgery show partial, heterogeneous reperfusion (arrows) and heterogeneously elevated ADC.

 

Figure 2
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  		Figure 2c: Top row: Coronal T1-weighted spin-echo (500/9) MR images obtained after administration of gadolinium-based MR contrast material. Bottom row: Line-scan diffusion MR imaging (2100/70, b = 0 and 700 mm2/sec) ADC maps. (a) Images of control hip 1 week after surgery show normal enhancement and diffusion. (b) Images of ischemic hip 1 week after surgery show complete loss of blood flow and increased ADC to femoral head. (c) Images of ischemic hip 2 weeks after surgery show partial, heterogeneous reperfusion (arrows) and heterogeneously elevated ADC.

 

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  		Comparison of ERs and ADCs of Ischemic Hips and Control Hips of Eight Piglets after Surgery

 

Figure 3
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  		Figure 3a: Graphs of (a) ER and (b) ADC time curves. Significant differences between ischemic and control hip values at given time point are indicated (*). Diffusion and gadolinium enhancement follow different time courses. ER values of ischemic hips increase from near zero 48 hours after surgery to control hip values at 4 and 8 weeks. ADC values of ischemic hips significantly increase at 1 week when compared with those of control hips and stay elevated above control hip values for remainder of study, despite restoration of blood flow. ADC values of control hips decrease significantly during the study, presumably because of age-related changes in matrix content and structure.

 

Figure 3
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  		Figure 3b: Graphs of (a) ER and (b) ADC time curves. Significant differences between ischemic and control hip values at given time point are indicated (*). Diffusion and gadolinium enhancement follow different time courses. ER values of ischemic hips increase from near zero 48 hours after surgery to control hip values at 4 and 8 weeks. ADC values of ischemic hips significantly increase at 1 week when compared with those of control hips and stay elevated above control hip values for remainder of study, despite restoration of blood flow. ADC values of control hips decrease significantly during the study, presumably because of age-related changes in matrix content and structure.

 
ER values of the ischemic hips increased from 48 hours through 8 weeks after surgery, which reveals a large significant increase in this group (Greenhouse-Geisser F = 12.48, P = .007). Specific time-point comparisons revealed a sixfold increase between 1 week after surgery and 4 weeks after surgery (0.18 ± 0.16 vs 1.07 ± 0.51, P = .008); there was no significant difference in ER values of the control hips between 1 week after surgery and 4 weeks after surgery (P = .25).

Diffusion changes after surgery followed a different time course than did enhancement changes (Figs 1–3). ADC values were significantly higher for ischemic hips than for control hips at 1, 2, 4, and 8 weeks after surgery (P < .001), although no statistically significant difference was detected at 48 hours after surgery (P = .06). Therefore, ADC values were significantly higher for ischemic hips starting at 1 week after surgery and continuing throughout the study (Fig 3b) and remained elevated when compared with those of control hips, despite the onset of reperfusion.

ADC values of the ischemic hips were consistently high and were without any significant time-related differences (F = 1.62, P = .25). ADC values of the control hips declined over time (F = 17.92, P < .001).

Overall, SI changes due to ischemia on T1- and T2-weighted images were variable and did not parallel the changes observed on diffusion or gadolinium-enhanced MR images. On T1-weighted images, SI changes fluctuated from week to week; no pattern was identified. T1-weighted images showed signal characteristics of hematopoietic marrow in the control hips, which could have obscured SI changes related to ischemia. On T2-weighted images, the ischemic femoral epiphyses of four piglets had increased SI throughout the study (consistent with edema); in three other piglets, an initial SI decrease was followed by an increase after week 1; SI had a variable evolution in one piglet.

Anatomic Changes
Several anatomic changes were evident on MR images. After week 2, there was progressive broadening of the cartilaginous epiphysis, fragmentation and flattening of the secondary ossification center, and occasional premature closure of the physis. These changes coincided with the initiation of reperfusion (ie, after the elevation in ADC) and continued throughout the study without any reversal due to reperfusion. After the final imaging time point, the animals were sacrificed and hips were removed for evaluation. Results of gross examination revealed growth abnormalities of the ischemic proximal femoral head and neck in each animal. Shortening of the ischemic femoral head and neck compared with the control femoral head and neck, which indicated proximal femoral growth-plate dysfunction, ranged from 0.4 to 1.8 cm. Greater trochanter growth was always maintained, as expected, because the trochanter is supplied by a different vascular source. The shortened ischemic femoral head and neck with persisting normal trochanter growth led to coxa vara deformity. Figure 4 shows the ischemic and control hips in gross photographs (coronal plane hemisection) and specimen radiographs.


Figure 4
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  		Figure 4a: (a) Top: Photograph of gross section (4.2 cm) from control femoral head of typical piglet at end of 8-week study. Bottom: Photograph of gross section from ischemic femoral head of typical piglet at end of 8-week study. Control femoral head has normal, smooth, curved surface and demarcated structures (secondary ossification center, physis). Ischemic head is broader and flatter than control head. Note position of ligature (arrows) in cross-section. Epiphyseal cartilage is thicker in the ischemic femoral head than in control femoral head, and physis appears to be disrupted. A cartilaginous island has appeared in what was secondary ossification center. There appear to be marrow changes in epiphysis; there is more red marrow (less fatty marrow) in ischemic femoral head than in control femoral head; this is consistent with elevated diffusion values in ischemic femoral head because red marrow has higher diffusion than yellow marrow (20). (b) Radiograph of control (left) and ischemic (right) femurs of another piglet shows broadening, fragmentation, and flattening of ischemic femoral head (arrow) and shortening of femoral neck.

 

Figure 4
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  		Figure 4b: (a) Top: Photograph of gross section (4.2 cm) from control femoral head of typical piglet at end of 8-week study. Bottom: Photograph of gross section from ischemic femoral head of typical piglet at end of 8-week study. Control femoral head has normal, smooth, curved surface and demarcated structures (secondary ossification center, physis). Ischemic head is broader and flatter than control head. Note position of ligature (arrows) in cross-section. Epiphyseal cartilage is thicker in the ischemic femoral head than in control femoral head, and physis appears to be disrupted. A cartilaginous island has appeared in what was secondary ossification center. There appear to be marrow changes in epiphysis; there is more red marrow (less fatty marrow) in ischemic femoral head than in control femoral head; this is consistent with elevated diffusion values in ischemic femoral head because red marrow has higher diffusion than yellow marrow (20). (b) Radiograph of control (left) and ischemic (right) femurs of another piglet shows broadening, fragmentation, and flattening of ischemic femoral head (arrow) and shortening of femoral neck.

 
Histologic Changes
Histologic differences (Fig 5) were noted between control (Fig 5a) and ischemic femoral heads (Fig 5b, 5c). Thickening and hypercellularity of the ischemic epiphyseal cartilage were observed (Fig 5b). The surface articular cartilage was invariably misshapen and often had areas of chondrocyte cloning, chondrocyte death, and fibrous tissue proliferation. In addition, there were numerous cartilaginous excursions extending into the secondary ossification center, which indicated arrest of the normal process of endochondral ossification (Fig 5b). The ischemic femoral heads had undergone revascularization and repair, but also considerable microstructural disorganization, which included evidence of persisting fibrous tissue proliferation, new bone formation by both endochondral and direct intramembranous mechanisms, and variable persistence of fibro-osseous and chondro-osseous tissues not seen in normal development. More confirmation of growth disturbance was found in the form of regions of focal growth plate disruption. Results of histologic examination also revealed that reperfusion of the ischemic femoral head occurred through new vessels (as opposed to through the surgically ligated vessel originally supplying the head) that grew over and around the ligature to penetrate the head. Vascularization of the epiphyseal cartilage was frequently greater than normal.


Figure 5
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  		Figure 5a: Photomicrographs 8 weeks after surgery of (a) control femoral head and (b, c) ischemic femoral head. (a) Photomicrograph of entire control femoral head (midcoronal plane). Surface is curvilinear, and cartilage is of uniform thickness. Endochondral bone has formed on undersurface of epiphyseal cartilage, and bony trabeculae are dispersed throughout secondary ossification center. (b) Photomicrograph of entire ischemic femoral head. Surface of articular cartilage is misshapen. Articular and epiphyseal cartilages are thicker than in control femoral head, and undersurface of epiphyseal cartilage is irregular. Although endochondral bone is forming, a wedge of epiphyseal cartilage is persisting centrally into secondary ossification center (arrow). Bony trabeculae are irregularly dispersed in secondary ossification center. Fibrous tissue predominates above physis on right. (c) Higher-power photomicrograph shows repair tissue within ischemic femoral head that is indicative of revascularization but is highly irregular pattern of tissue types. Endochondral bone has formed below epiphyseal cartilage (E), and physeal cartilage (P) is intact. However, there is abnormal persisting cartilage (left), fibro-osseous tissue (right), and intramembranous bone above physis. (Toluidine blue stain; original magnification, x10.)

 

Figure 5
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  		Figure 5b: Photomicrographs 8 weeks after surgery of (a) control femoral head and (b, c) ischemic femoral head. (a) Photomicrograph of entire control femoral head (midcoronal plane). Surface is curvilinear, and cartilage is of uniform thickness. Endochondral bone has formed on undersurface of epiphyseal cartilage, and bony trabeculae are dispersed throughout secondary ossification center. (b) Photomicrograph of entire ischemic femoral head. Surface of articular cartilage is misshapen. Articular and epiphyseal cartilages are thicker than in control femoral head, and undersurface of epiphyseal cartilage is irregular. Although endochondral bone is forming, a wedge of epiphyseal cartilage is persisting centrally into secondary ossification center (arrow). Bony trabeculae are irregularly dispersed in secondary ossification center. Fibrous tissue predominates above physis on right. (c) Higher-power photomicrograph shows repair tissue within ischemic femoral head that is indicative of revascularization but is highly irregular pattern of tissue types. Endochondral bone has formed below epiphyseal cartilage (E), and physeal cartilage (P) is intact. However, there is abnormal persisting cartilage (left), fibro-osseous tissue (right), and intramembranous bone above physis. (Toluidine blue stain; original magnification, x10.)

 

Figure 5
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  		Figure 5c: Photomicrographs 8 weeks after surgery of (a) control femoral head and (b, c) ischemic femoral head. (a) Photomicrograph of entire control femoral head (midcoronal plane). Surface is curvilinear, and cartilage is of uniform thickness. Endochondral bone has formed on undersurface of epiphyseal cartilage, and bony trabeculae are dispersed throughout secondary ossification center. (b) Photomicrograph of entire ischemic femoral head. Surface of articular cartilage is misshapen. Articular and epiphyseal cartilages are thicker than in control femoral head, and undersurface of epiphyseal cartilage is irregular. Although endochondral bone is forming, a wedge of epiphyseal cartilage is persisting centrally into secondary ossification center (arrow). Bony trabeculae are irregularly dispersed in secondary ossification center. Fibrous tissue predominates above physis on right. (c) Higher-power photomicrograph shows repair tissue within ischemic femoral head that is indicative of revascularization but is highly irregular pattern of tissue types. Endochondral bone has formed below epiphyseal cartilage (E), and physeal cartilage (P) is intact. However, there is abnormal persisting cartilage (left), fibro-osseous tissue (right), and intramembranous bone above physis. (Toluidine blue stain; original magnification, x10.)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 References
 
Results of our study show that in epiphyseal ischemia, diffusion increases early and remains elevated despite reperfusion. Although the initial insult can be depicted with gadolinium-enhanced imaging, the subsequent reperfusion such imaging depicts is not predictive of recovery and could instead accelerate epiphyseal destruction.

The changes in diffusion in the control hips have to be interpreted in the context of normal growth and development. The observed age-related decreases in diffusion (ie, changes in ADC values of control hips) are presumably because of the normal changes in ossification, marrow transformation, and matrix content that occur in developing epiphyses due to skeletal development. Thus, changes in this and other MR parameters (21) in the growing skeleton do not necessarily indicate disease but may instead reflect normal maturation. This finding implies that diffusion values need to be indexed to age-appropriate values to assess ischemic changes. Alternatively, this parameter could be used to estimate skeletal maturation in the absence of ischemia.

Earlier work has shown that diffusion is elevated in acute skeletal ischemia (24–72 hours) in animals (8). To our knowledge, ours is the first study to assess the value of diffusion-weighted MR imaging in prolonged skeletal ischemia by using an animal model and lasting impairment of the blood supply that leads to necrosis, which closely parallels human disease. This model has been shown to result in epiphyseal changes that, at gross morphologic and histologic examination, closely resemble human avascular necrosis seen in conditions such as Legg-Calvé-Perthes disease (22–25).

We found that diffusion changes follow a different time course than do enhancement changes. Ischemic diffusion values were higher than those of control diffusion values shortly after the induction of ischemia and before the initiation of reperfusion or the manifestation of gross anatomic changes. This finding suggests that the diffusion increase reflects damage due to the initial ischemic insult rather than reperfusion or injury caused by reperfusion. The compensatory reperfusion did not avert the progressive destruction of the femoral head. Instead, tissue structural abnormalities persisted after reperfusion and appeared to accelerate after blood flow was restored. Diffusion remained elevated despite restoration of blood flow. The thickening of epiphyseal cartilage and the presence of cartilaginous islands within ossified tissue seen at histologic findings are consistent with elevated diffusion because cartilage has higher diffusion values than bone.

Diffusion changes may be a better indicator of lasting femoral head damage than are enhancement changes and may precede gross deformity. Reperfusion was initiated too late to reverse or minimize damage and/or it contributed to tissue destruction as well as tissue repair, although in a disorganized fashion. We suspect that this damage may be because of reperfusion occurring spontaneously and nonuniformly in this animal model and speculate that flow restoration at an earlier time point would instead preserve the structure of the femoral head (26). Further studies are needed to evaluate whether ischemic diffusion values will return to the level of control diffusion values if flow is restored in a more timely and uniform manner.

The main limitation of this study was that the ischemic epiphysis follows a relentless course to epiphyseal destruction in this model. In Legg-Calvé-Perthes disease and other types of epiphyseal ischemia, epiphyseal damage is variable, and tissue healing often occurs. Further evaluation of the prognostic value of diffusion requires a model in which the insult is transient or incomplete and the outcome is variable. Investigation of diffusion changes at more distant time points also would be useful. High-spatial-resolution imaging of the epiphysis, however, becomes difficult as piglets grow because of the increased imaging time required to maintain signal-to-noise ratio when distance from the surface coil is increased and because of the increased field of view needed to image larger animals. Although we did not directly measure perfusion in this study, we believe that enhancement changes are a result of perfusion changes. Besides perfusion abnormalities, however, blood volume and vascular permeability abnormalities also affect enhancement.

We have shown that diffusion values can remain abnormally elevated despite flow restoration to the femoral head after prolonged ischemia; this finding suggests that diffusion MR imaging may be of value in determining the timing and efficacy of treatment options to restore vascularity. The combination of the two MR imaging techniques provides an interesting view into the evolving pathobiology of skeletal ischemia.

Practical application: Diffusion MR images of the immature ischemic epiphysis can be obtained reliably. The information obtained from diffusion values precedes morphologic changes and is a better marker of epiphyseal destruction than are enhancement changes. Therefore, MR diffusion imaging is complementary to conventional and gadolinium-enhanced MR imaging in epiphyseal ischemia.


   ADVANCES IN KNOWLEDGE
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
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   FOOTNOTES
 

Abbreviations: ADC = apparent diffusion coefficient • ER = enhancement ratio • SI = signal intensity

2 Current address: Department of Radiology, Children's Hospital of Philadelphia, Philadelphia, Pa Back

Authors stated no financial relationship to disclose.

Author contributions: Guarantor of integrity of entire study, D.J.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; approval of final version of submitted manuscript, all authors; literature research, N.M.M., S.A.C., F.S., E.A.O., D.J.; experimental studies, N.M.M., S.A.C., F.S., E.A.O., R.M.J., D.J.; statistical analysis, N.M.M., D.Z.; and manuscript editing, N.M.M., S.A.C., F.S., E.A.O., D.J.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 

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