OARSI 2011
1
QUANTIFICATION OF TRABECULAR BONE VASCULARITY IN A RABBIT MODEL OF OSTEOARTHRITIS BY PERFUSION MICRO COMPUTED TOMOGRAPHY D. Lickorish , M.M. Haskell , S.A. Wood, A. Collett, M.E.P. Goad, D. Schrier, C.M. Flory Grafton, MA, USA Introduction Results Objective Conclusions Reference To determine if a barium/gelatin perfusion protocol can yield a reproducible method of quantifying blood vessel architecture in the distal femur of a rabbit following ACLT/MMT The rabbit ACLT/MMT model is a common model of post-traumatic OA, yet no data exists describing the 3D vascular anatomy within the femoral subchondral trabecular space. Intravascular perfusion of heavy metal preparations combined with microCT has found increasing use as a method of analysis of vascular architecture. Characterization of the vascularity of the distal femur in the ACLT/MMT model of OA may shed light on vascular changes that occur following ACLT trauma and provide an additional analytical endpoint for pharmaceutical interventional studies. Fig. 8: Vascularity of the distal trabecular bony compartments as described by Fig.2 Density and caliber of vessels are larger in the ACLT/MMT joint (A) and extend towards the subchondral bone plate with numerous interconnections. Vessels of the unoperated control (B), in contrast, were smaller with fewer interconnections. Entry of vessels into the distal trabecular compartment was primarily from the posterior intercondylar region with occasional contributions through the anteromedial and anterolateral walls of the femoral condyles. (Bar = 5mm) Fig.3: Complete perfusion using 30% (w/v) barium suspended in 2% gelatin was achieved. No extravasation of the barium/gelatin mixture was observed histologically. Perfusate remained within the vasculature of the bone marrow (A) , distal trabecular bone (B) and subchondral bone plate (C) of the femur. Limitations of microCT scan resolution prevent practical quantitative analysis of vessels below 40μm in diameter. A C B Fig.6: Lateral MicroCT radiographs showing barium perfusion in ACLT/MMT (A) and unoperated contralateral control (B) . Posterior tibial artery is prominent Fig.5: 3D rendering of ACLT/MMT femur (left) showing subchondral bony changes (osteophytes) commonly seen in this model. Osteophytes are predominantly observed along the length of the medial femoral condyle. Contrast this with the smooth osseous surface of the contralateral control (right). Note foramina (arrow) in the posterior intercondylar area, a principal site of blood vessel entry. MicroCT data Distal Femur: Vessel Volume/Tissue Volume (%) Animal Left Femur - ACLT/MMT Right Femur - Contralateral Control 1 0.06 0.04 2 0.19 0.08 3 0.25 0.10 4 0.13 0.07 Perfusion Protocol (Adapted from Sider et al 2010) Female NZW rabbits (n=4) underwent ACLT/MMT surgery in the left stifle. At 8 weeks post-surgery animals were anesthetized using isofluorane and given 1000U of heparin by ear vein. A midline laparotomy and blunt dissection exposed the abdominal aorta and inferior vena cava (IVC) and 2 silk sutures were placed around each vessel approximately 1cm apart to ensure closure of vessels following perfusion (Fig. 1). A 16G catheter was introduced into the abdominal aorta in a caudal direction between the 2 sutures and securely tied in place with the distal suture. Prior to perfusion, the IVC was lacerated between the sutures to allow outflow. 300mls of warmed PBS with papaverine hydrochloride (0.02mg/mL) was perfused at a rate of 50mls/min followed by 250mls of a warmed solution of barium sulfate (30%w/v) in 2% gelatin until white venous outflow was observed. Ligatures were tied and the catheter removed. Animals were placed at 4 degrees overnight to allow gelatin to set. Fig. 1: Ligatures around the IVC (top) and abdominal aorta (bottom) prior to perfusion MicroCT Protocol Disarticulated femurs were formalin fixed, partially decalcified and scanned at 18μm resolution using a Skyscan 1076 Invivo microCT (88kv, 112mA, Al filter, Step 0.6º, 180 degree, 15min scan time). Medial collateral ligament insertion site (arrow) was used as an anatomic landmark for creation of ROI in the medial and lateral trabecular compartments of the distal femur (Fig. 2). ROI were manually drawn using CTAn to generate VOI for measurement. Vasculature was threshold segmented using a constant threshold level. Percent vessel volume/tissue volume (%VV/TV) was calculated for both ACLT/MMT and contralateral control femurs. Data were analyzed using paired t test. Samples were then prepared for conventional wax histology Fig. 2: ROI for distal vascular analysis Fig.4: Quantitative analysis by microCT yielded a consistent, significant increase in vascular volume in the ACLT/MMT femurs when compared to contralateral unoperated controls. Sider et al (2010) A new bone vascular perfusion compound for the simultaneous analysis of bone and vasculature, Microsc Res Tech. 73(7):665-72 Fig. 7: Posterior 3D rendering of data from Fig. 6 clearly demonstrates prolific soft tissue vascularity in ACLT/MMT (A) when compared to unoperated contralateral control (B) . F=Femur; T=Tibia A - Left ACLT/MMT B – Right Control • Protocol resulted in complete perfusion of distal rabbit limbs • Simple, rapid and reproducible technique • At 8wks, ACLT/MMT results in two-fold increase in vascularity over contralateral control limb • Suitable tool for quantitative analysis of vascular changes in preclinical models of OA. Ant. Ant. A - Left ACLT/MMT: Soft Tissue Vacularity F T F T LFC B - Right Control: Soft Tissue Vascularity B - Right Control: Intra-osseous Vascularity A - Left ACLT/MMT: Intra-osseous Vascularity MFC LFC MFC
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Transcript of OARSI 2011
QUANTIFICATION OF TRABECULAR BONE VASCULARITY IN A RABBIT MODEL OF OSTEOARTHRITIS BY PERFUSION MICRO COMPUTED TOMOGRAPHY
D. Lickorish, M.M. Haskell , S.A. Wood, A. Collett, M.E.P. Goad, D. Schrier, C.M. FloryGrafton, MA, USA
Introduction
Results
Objective
Conclusions
Reference
To determine i f a barium/gelatin perfusion protocol can yield a reproducible method of quantify ing blood vessel architecture in the distal femur of a rabbit fol lowing ACLT/MMT
The rabbit ACLT/MMT model is a common model of post -traumatic OA, yet no data exists describing the 3D vascular anatomy within the femoral subchondraltrabecular space. Intravascular perfusion of heavy metal preparations combined with microCT has found increasing use as a method of analysis of vascular architecture. Characterizat ion of the vascular ity of the distal femur in the ACLT/MMT model of OA may shed l ight on vascular changes that occur fol lowing ACLT trauma and provide an addit ional analyt ical endpoint for pharmaceutical interventional studies.
F ig . 8 : Vascular i ty of the d ista l t rabecular bony compartments as descr ibed by F ig .2 Dens i ty and ca l iber of vesse ls are larger in the ACLT/MMT jo int (A) and extend towards the subchondra l bone p late with numerous interconnect ions . Vesse ls of the unoperated contro l (B) , in contrast , were smal ler with fewer interconnect ions . Entry of vesse ls into the d ista l t rabecular compartment was pr imar i ly f rom the poster ior intercondylar reg ion with occas iona l contr ibut ions through the anteromedia l and antero latera l wal l s o f the femora l condyles . (Bar = 5mm)
F ig .3 : Complete perfus ion us ing 30% (w/v) bar ium suspended in 2% ge lat in was achieved. No extravasat ion of the bar ium/gelat in mixture was obser ved h isto log ica l ly. Perfusateremained with in the vasculature of the bone marrow (A) , d ista l t rabecular bone (B) and subchondra l bone p late (C) of the femur. L imitat ions of microCT scan reso lut ion prevent pract ica l quant i tat ive ana lys is o f vesse ls be low 40µm in d iameter.
A CB
F ig .6 : Latera l MicroCT radiographs showing bar ium perfus ion in ACLT/MMT (A)and unoperated contra latera l contro l (B) . Poster ior t ib ia l artery i s prominent
F ig .5 : 3D render ing of ACLT/MMT femur ( lef t ) showing subchondra l bony changes ( osteophytes ) commonly seen in th is model . Osteophytes are predominant ly obser ved a long the length of the media l femora l condyle . Contrast th is with the smooth osseous surface of the contra latera l contro l ( r ight ) . Note foramina (arrow) in the poster ior intercondylar area , a pr inc ipa l s i te of b lood vesse l entry.
MicroCT data
Distal Femur: Vessel Volume/Tissue Volume (%)
Animal Left Femur - ACLT/MMT Right Femur - Contralateral Control
1 0.06 0.04
2 0.19 0.08
3 0.25 0.10
4 0.13 0.07
Perfusion Protocol (Adapted from Sider et al 2010)
Female NZW rabbits (n=4) underwent ACLT/MMT surgery in the left st i f le. At 8 weeks post-surgery animals were anesthetized using isof luorane and given 1000U of heparin by ear vein. A midl ine laparotomy and blunt dissection exposed the abdominal aorta and inferior vena cava ( IVC) and 2 s i lk sutures were placed around each vessel approximately 1cm apart to ensure c losure of vessels fol lowing perfusion (F ig. 1) .
A 16G catheter was introduced into the abdominal aorta in a caudal direction between the 2 sutures and securely t ied in place with the distal suture. Pr ior to perfusion, the IVC was lacerated between the sutures to al low outf low. 300mls of warmed PBS with papaverine hydrochloride (0.02mg /mL) was perfused at a rate of 50mls/min fol lowed by 250mls of a warmed solution of barium sulfate (30%w/v) in 2% gelat in unti l white venous outf low was observed. L igatures were t ied and the catheter removed. Animals were placed at 4 degrees overnight to al low gelat in to set.
F i g . 1 : L i ga t u re s a ro u n d t h e I VC ( t o p ) a n d a b d o m i n a l a o r t a ( b o tt o m ) p r i o r t o p e r f u s i o n
MicroCT ProtocolDisart iculated femurs were formalin f ixed, part ia l ly decalc if ied and scanned at 18µm resolution using a Skyscan 1076 Invivo microCT (88kv, 112mA, Al f i l ter, Step 0.6º, 180 degree, 15min scan t ime). Medial col lateral l igament insert ion s ite (arrow) was used as an anatomic landmark for creation of ROI in the medial and lateral trabecularcompartments of the distal femur (F ig. 2) . ROI were manual ly drawn using CTAn to generate VOI for measurement.
Vasculature was threshold segmented using a constant threshold level . Percent vessel volume/tissue volume (%VV/TV) was calculated for both ACLT/MMT and contralateralcontrol femurs. Data were analyzed using paired t test . Samples were then prepared for conventional wax histology
F i g . 2 : R O I fo r d i s t a l va s c u l a r a n a l y s i s
F ig .4 : Quant i tat ive ana lys is by microCTyie lded a cons istent , s ign i f icant increase in vascular vo lume in the ACLT/MMT femurs when compared to contra latera lunoperated contro ls . Sider et al (2010) A new bone vascular perfusion compound for the simultaneous analysis of bone and vasculature,
Microsc Res Tech. 73(7):665-72
F ig . 7 : Poster ior 3D render ing of data f rom F ig . 6 c lear ly demonstrates pro l i f i c sof t t i ssue vascular i ty in ACLT/MMT (A) when compared to unoperatedcontra latera l contro l (B) . F=Femur; T=T ib ia
A - Left ACLT/MMT B – Right Control
• Protocol resulted in complete perfusion of distal rabbit limbs • Simple, rapid and reproducible technique• At 8wks, ACLT/MMT results in two-fold increase in vascularity over contralateral control limb• Suitable tool for quantitative analysis of vascular changes in preclinical models of OA.
Ant. Ant.
A - Left ACLT/MMT: Soft Tissue Vacularity
F
T
F
T
LFC
B - Right Control: Soft Tissue Vascularity
B - Right Control: Intra-osseous VascularityA - Left ACLT/MMT: Intra-osseous Vascularity
MFCLFC MFC
