Pilot study for detection of early changes in tissue associated with heterotopic ossification: moving toward clinical use of Raman spectroscopy

<div><p></p><p>Over 60% of combat-wounded patients develop heterotopic ossification (HO). Nearly 33% of them require surgical excision for symptomatic lesions, a procedure that is both fraught with complications and can delay or regress functional rehabilitation. Relative medical contraindications limit widespread use of conventional means of primary prophylaxis, such as nonspecific nonsteroidal anti-inflammatory medications and radiotherapy. Better methods for risk stratification are needed to both mitigate the risk of current means of primary prophylaxis as well as to evaluate novel preventive strategies currently in development. We asked whether Raman spectral changes, measured <i>ex vivo</i>, could be associated with histologic evidence of the earliest signs of HO formation and substance P (SP) expression in tissue biopsies from the wounds of combat casualties. In this pilot study, we compared normal muscle tissue, injured muscle tissue, very early HO lesions ( < 16 d post-injury), early HO lesions ( > 16 d post-injury) and mature HO lesions. The Raman spectra of these tissues demonstrate clear differences in the Amide I and III spectral regions of HO lesions compared to normal tissue, denoted by changes in the Amide I band center (<i>p</i> < 0.01) and the 1340/1270 cm⁻¹ (<i>p</i> < 0.05) band area and band height ratios. SP expression in the HO lesions appears to peak between 16 and 30 d post-injury, as determined by SP immunohistochemistry of corresponding tissue sections, potentially indicating optimal timing for administration of therapeutics. Raman spectroscopy may therefore prove a useful, non-invasive and early diagnostic modality to detect HO formation before it becomes evident either clinically or radiographically.</p></div

Functional outcomes in sham and experimental ischemia groups.

<p>A) Mean drop foot scores for all experimental groups. A drop foot score of 0 meant that no neuropathy was observed post-operatively. A drop foot score of 1 was consistent with neuropathy in the affected limb for at least one day post-operatively. A drop foot score of 2 was consistent with neuropathy in the affected limb for at least three days post-operatively. A drop foot score of 3 was indicative of neuropathy in the affected limb beyond 5 days post-operatively. B) Mean Tarlov scores for all experimental groups. The dashed line at 5.0 is reflective of normal locomotion. The asterisks indicate statistically significant differences when compared to sham and 3.5 hour occlusion animals—** = p-value < 0.01, *** = p-value < 0.001.</p

Slopes of reperfusion and post-occlusive reactive hyperemia for 3CCD and IR imaging.

<p>Bar plots comparing the IR slopes of reperfusion for all experimental groups (A) and 3CCD slopes of reperfusion, slopes of PORH at 5 minutes reperfusion, and slopes of PORH at 10 minutes reperfusion for all experimental groups (B). Statistically significant differences between 4.7 hour tourniquet animals and other experimental groups are designated by an asterisk (*)–p-value<0.05. Note, for each set of slopes presented in (B), the data is plotted by lowest Tarlov score (i.e. least normal locomotion) and increases to the highest Tarlov score (i.e. most normal locomotion). 3_5O = 3.5 hour occlusion; 3_5T = 3.5 hour tourniquet; 4_7O = 4.7 hour occlusion; 4_7T = 4.7 hour tourniquet.</p

3CCD and infrared (IR) imaging. Top) Representative grayscale 3CCD images of a hind limb, and Middle) representative grayscale infrared images of a hind limb at (1) baseline, (2) maximum ischemia, (3) 10 minutes post-reperfusion, and (4) 30 minutes post-reperfusion.

<p>Bottom left) Profile of R-B values, derived from 3CCD imaging, over the course of 3.5 hours of ischemia and 30 minutes of reperfusion. Bottom right) Profile of IR values and corresponding mean leg temperature over the course of 3.5 hours of ischemia and 30 minutes of reperfusion. The time points mentioned previously (1–4) are noted.</p

Profiles of operative 3CCD and IR imaging values.

<p>Bar plots of mean R-B values derived from 3CCD imaging (A) and mean leg temperatures in °F (B) for all experimental groups at baseline, maximum ischemia, 10 minutes post-reperfusion, and 30 minutes post-reperfusion. Error bars = SEM (standard error of the mean). 3_5O = 3.5 hour occlusion; 3_5T = 3.5 hour tourniquet; 4_7O = 4.7 hour occlusion; 4_7T = 4.7 hour tourniquet.</p

Outcome based comparison of various metrics.

<p>Values are means +/- the standard deviation for animals that 1) recovered full locomotion, and 2) did not recover full locomotion. Differences approaching statistical significance (p-value ≈ 0.1) are indicated by (†). Statistically significant differences (p-value<0.05) are indicated by an asterisk (*).</p

RT-PCR targets with primer and probe sequences.

<p>RT-PCR targets with primer and probe sequences.</p

Peripheral leukocyte counts during hemorrhage and reperfusion periods.

<p>Dashed line indicates end of hemorrhage and beginning of reperfusion period. Leukocytes were significantly decreased during the reperfusion period in the FTY720 group (p = 0.03). Data is depicted as mean ± SEM.</p

PLSDA models to predict return to normal locomotion for all data, imaging only data, and non-imaging data sets.

<p>Receiver operating curves for both calibration and cross-validation data sets. Area under the curve (AUC), a surrogate for accuracy, is displayed for each curve. A) PLSDA model generated from all parameters, but excluding outcome parameters. B) PLSDA model calculated from only imaging parameters. C) PLSDA model generated from all non-imaging parameters, but excluding outcome parameters. The calibration data is plotted in black and the cross-validation data is plotted in gray.</p

Experimental design.

<p>Liver injury was initiated at t = 0. Uncontrolled hemorrhage occurred until 1 hour, at which time the abdomen was packed and temporarily closed (pre-hospital phase). The animal was observed until 2 hours when hospital care was initiated. The liver was repaired and the abdomen definitively closed. The animal was then observed for a total of 72 hours. Blood transfusion was administered as indicated. Necropsy was performed when the animal expired or at 72 hours following euthanasia. The uncontrolled hemorrhage and pre-hospital phases were considered the hemorrhage period, while the hospital care phase was considered the reperfusion period. In the experimental group, FTY720 (0.3 mg/kg diluted in 250 mL of NS)) was administered 15 minutes following liver injury. Vehicle controls were treated with 250 mL of NS.</p