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MUSA - Alberta Pharmacy Students' Association

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it has been found that PPAR-γ agonists are<br />

involved in vascular biology, inflammatory<br />

responses, tissue repair, cell differentiation<br />

and proliferation. 12-15 In arthritic<br />

synoviocytes, PPAR-γ agonists greatly<br />

inhibit inflammatory cytokine expression. 16<br />

PPAR-γ agonists have also been expressed in<br />

human chondrocytes acting as a protective<br />

17, 18<br />

mechanism for cartilage.<br />

PPAR-γ agonists have therapeutic potential<br />

in a variety of clinical conditions. There is<br />

literature showing PPAR-γ agonists role in<br />

fat metabolism and their anti-inflammatory<br />

actions shown through their effect on<br />

inhibiting cytokine expression. 19, 20 There<br />

are limited published studies looking at the<br />

effect of PPAR-γ agonists in osteoarthritis<br />

models. One study by Kobayashi et al.,<br />

using a partial medial menisectomy<br />

guinea pig model, found that the PPAR-γ<br />

agonist pioglitazone reduced cartilage<br />

lesion depth and area. 21 By studying the<br />

effect of the PPAR-γ agonist rosiglitazone<br />

on the adaptive physiological response<br />

in ACL ruptured knees (specifically the<br />

angiogenic activity), its potential as a new<br />

therapy to treat osteoarthritic patients can<br />

be determined.<br />

The purpose of this study is to examine the<br />

effect of the PPAR-γ agonist rosiglitazone on<br />

the physiological and angiogenic responses<br />

in the rabbit model of joint laxity and<br />

osteoarthritis. The rabbit model was chosen<br />

as we can compare the results obtained<br />

to our established database on adaptive<br />

physiology in rabbit ACL – ruptured joints.<br />

It is hypothesized that PPAR-γ agonists<br />

will decrease physiological degeneration<br />

of the MCL in the ACL ruptured knee.<br />

The blood vessel volume and degree of<br />

angiogenesis occurring in the MCL of the<br />

ACL ruptured knees will be measured using<br />

immunohistochemistry for specific markers<br />

for vascular endothelium (CD-31) and<br />

vascular smooth muscle (SMA) in the MCL.<br />

MATERIALS AND METHODS<br />

Subjects<br />

Six young skeletally mature 1-year-old New<br />

Zealand white rabbits (4.5 - 6.5kg; Riemans<br />

Fur Ranch, St. Agatha, Ontario) were<br />

assigned to one of three groups: control<br />

(n=2); 4-week right leg ACL transected<br />

(ACL-X) (n=2); 4-week right leg ACL-X<br />

treated with a low dose of 5 mg/kg /day of<br />

rosiglitazone (n=2). The two contralateral<br />

MCLs in the 4-week ACL-X rabbits treated<br />

with rosiglitazone were used as the drug<br />

treated non-ACL ruptured leg controls. This<br />

results in a total of 8 MCLs analyzed in this<br />

study. Rabbits were kept on a 12 – hour<br />

light/dark cycle and fed standard laboratory<br />

chow and tap water ad libitum. All animals<br />

were treated and maintained according<br />

to the Canadian Council on Animal Care<br />

guidelines and this study received approval<br />

by the University of Calgary Faculty of<br />

Medicine Animal Care Committee.<br />

aCL transection and<br />

Rosiglitazone injections<br />

Rabbits were given 0.18 mL of acepromazine<br />

maleate (Atravet ®) intravenously and<br />

anesthetized with halothane (2-5%, 1.0 L/<br />

min O ). All ACL transection surgeries were<br />

2<br />

completed on the right leg of the rabbits.<br />

An anterior tibial draw test was performed<br />

on the right leg to ensure no prior ACL<br />

injury existed. The anterior tibial draw test<br />

was done by grasping the tibia with both<br />

hands below the joint line, thumbs placed<br />

on either side of the patella, with the tibia<br />

pulled anteriorly.<br />

An antero-lateral surgical approach was<br />

used. The ligament was exposed by lateral<br />

subluxation of the patella and reflection<br />

of the intra-articular fat pad. The ACL was<br />

isolated using a hooked probe and the<br />

ligament was transected at the middle with<br />

a #12 hooked blade. A second anterior<br />

tibial draw was performed to ensure the<br />

transection was complete. Following the<br />

unilateral surgery, rabbits were treated with<br />

standard antibiotics and allowed to resume<br />

normal cage activity for four weeks.<br />

Rosiglitazone treated animals were injected<br />

subcutaneously with a low dose of 5 mg/<br />

kg of body weight per day for a total of four<br />

weeks. In the ACL-X rabbits, injections<br />

began on the day following the ACL<br />

transection surgery. Since the contralateral<br />

MCL was used as the non-operated leg<br />

rosiglitazone treated control, the 4-week<br />

drug treatment was simultaneous. At the<br />

beginning and end of the 4-week dosing<br />

period, anterior tibial draw tests were<br />

completed on the left leg to ensure no ACL<br />

injury existed.<br />

Immunohistochemistry<br />

MCLs were sectioned and labeled for CD-31<br />

and SMA according to the following doublelabel<br />

protocol. Rabbits were euthanized then<br />

the MCLs were harvested from control, ACL<br />

ruptured and rosiglitazone treated knees<br />

then cleaned of any extra tissue. Tissues were<br />

cryopreserved in serial sucrose solutions<br />

of 10%, 20% and 30% concentrations.<br />

Following cryoprotection, ligaments were<br />

frozen in isopentane at -80°C, embedded<br />

in OCT media then stored at -30°C for one<br />

month until processing. MCLs were cut into<br />

100 μm thick longitudinal serial sections and<br />

placed individually in 24 well plastic plates.<br />

Sections were washed in phosphate buffered<br />

saline (PBS) (3 x 10 minutes) then immersed<br />

in 10% normal donkey serum (Jackson<br />

Immunoresearch, West Grove, PA, USA)/<br />

PBS 1% Triton X100 for 1 hour at room<br />

temperature. MCLs were then incubated<br />

with mouse anti-human CD-31 antibody<br />

(1:50 dilution; Dako, Carpinteria, CA, USA)<br />

for 24 hours at 4°C in a humidified chamber.<br />

Sections were washed in PBS (3 x 10<br />

minutes) and incubated with donkey antimouse<br />

Cy5 conjugated secondary antibody<br />

(1:300 dilution; Jackson Immunoresearch,<br />

West Grove, PA, USA) for 2 hours at room<br />

temperature. Sections were washed in<br />

PBS (3 x 10 minutes) then incubated with<br />

goat anti-human smooth muscle actin<br />

(SMA) antibody (1:400 dilution, Novus<br />

Biologicals, Littleton, CO, USA) for 24 hours<br />

at 4°C, followed by further PBS washes<br />

and incubation with donkey anti-goat Cy2<br />

conjugated antibody (1:400 dilution; Jackson<br />

Immunoresearch, West Grove, PA, USA)<br />

for 2 hours at room temperature. Sections<br />

were then washed for a final time, placed<br />

on slides with Fluorsave TM (Calbiochem,<br />

Mississauga, Ontario) and coverslipped.<br />

Slides were stored in a cardboard slide<br />

holder to protect fluorescence loss due<br />

to light.<br />

Confocal Microscopy<br />

MCL sections were analyzed using an<br />

Olympus Fluoview FV-1000 confocal<br />

microscope. They were visualized under a<br />

10x objective and imaged using 4-micron<br />

thick optical z-stack sections. Simultaneous<br />

dual channel scanning laser confocal<br />

analysis was performed using preconfigured<br />

Cy2 and Cy5 channel settings. Images were<br />

saved in the OIF file format.<br />

Statistical analysis<br />

The CD-31 and SMA volumes found in<br />

the MCL were determined using Image<br />

J software. The volumes of CD-31 and<br />

SMA in each image were put into an excel<br />

spreadsheet and then summed together to<br />

get a total volume converted into milliliters<br />

for each ligament. The volumes of CD-31<br />

and SMA for each MCL type had their<br />

averages calculated. Since there are only two<br />

subjects per group, the mean and the range<br />

of data was the chosen method of statistical<br />

analysis. If the subjects in each group had<br />

increased numbers, inferential analysis<br />

using a two – way ANOVA would have been<br />

preferred for comparative purposes. By using<br />

this method of analysis, one could then<br />

interpret if rosiglitazone has an effect on the<br />

angiogenic response in this pilot study.<br />

University of <strong>Alberta</strong> Health Sciences Journal • April 2012 • Volume 7 • Issue 1 5<br />

RESEARCH

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