mvc view to pdf itextsharp: Adding text to pdf file SDK control service wpf azure web page dnn 92015006322-part1593

normal tissue remodelling [12] and cause excessive tibial

femoral compressive force [10].

The present study showed that severe graft degeneration

at the intra-articular mid-substance may contribute to the

lower knee stiffness at 2 weeks post-operation. As graft

degradation is presumably mediated by inﬁltrating healing

cells, we speculate that the tension-induced changes in col-

lagen ﬁbrillar ultrastructure[6] andthetissuereorganization

associated with Poisson contraction [13] may retard the

inﬁltrationof healingcellsandbetter preservegraftintegrity.

At 6 weeks post-operation, signiﬁcant graft remodelling

(ligamentization) occurred and the effect of initial graft

tensioning became insigniﬁcant. It suggests that graft ten-

sioning may only affect early stages of graft healing.

In contrast to previous animal studies which used bone–

patellar tendon–bone (BPTB) grafts, this study investigates

the effect of graft tension on interface healing in ACLR

using free tendon graft. Although graft degeneration inside

femoral tunnel was similar in both tensioning groups, peri-

graft bone changes manifested as bone intrusions into graft

wereonlypresentin2 Nbutnotin 4 Ngroup(Fig.5).Initial

graft tensioning appeared to affect the graft-to-bone inter-

actions, and adverse peri-graftbone changes may negatively

affect the strength of graft-to-bone attachment in ACLR

[15].

Though the small size of rat may cause some technical

difﬁculties on the precision of the surgical procedures, rat

modelof ACLR wasstillusedbyotherresearchers[4]. Inour

study, the variations of failure parameters and laxity mea-

sures were not particularly large as compared to ACLR

studiesinlarger animals [1,8]. Our choiceof grafttension(2

or 4 N) corresponded to 5–10 % of ultimate strength of rat

ACL (*40 N), which is similar to the rangeused in clinical

practice (80–150 N) [3] thatcorrespondedto4–7 %of intact

human ACL (2,160 N) [16]. Thus, the use of a rat model is

still valuablefor investigation into initial grafttensioning in

ACLR. In our study, the measurement of A-P knee laxity

entailed larger random variations as compared to the knee

stiffness data, probably due to the variations in deﬁning

neutral position in knee laxity test. Further improvement in

standardizationof the neutralposition willbe favourable. As

6weeks in adult rat was roughly equivalent to 4 years in

human[14],themaximumobservationtimeinthis studywas

similar to the follow-up periodinprevious clinicalstudiesof

graft pre-tensioning [7, , 9, 11]. Yet, further study with a

longer follow-up may be necessary for studies of ligamen-

tization and chondral lesions.

Conclusion

In ACLR using free tendon graft, a higher initial graft

tension not only favoured the restoration of knee laxity, but

also enhanced graft incorporation into the bone tunnels in

early healing stage in the rat model. It suggests that early

mobilization will be safer for post-operative rehabilitation

in ACLR with a higher initial graft tension.

Acknowledgments This studywas supportedbythe Innovationand

Technology Fund (Ref: ITS/444/09) from the Innovation and Tech-

nology Commission of the government of Hong Kong SAR.

References

1. Abramowitch SD, Papageorgiou CD, Withrow JD, Gilbert TW,

Woo SL (2003) The effect of initial graft tension on the biome-

chanical properties of a healing ACL replacement graft: a study

in goats. J Orthop Res 21(4):708–715

2. AndersonK,Seneviratne AM,Izawa K, AtkinsonBL, Potter HG,

Rodeo SA (2001) Augmentation of tendon healing in an intra-

articular bone tunnel with use of a bone growth factor. Am J

Sports Med 29(6):689–698

3. Arneja S, McConkey MO, Mulpuri K, Chin P, Gilbart MK,

Regan WD, Leith JM (2009) Graft tensioninginanterior cruciate

ligament reconstruction: a systematic review of randomized

controlled trials. Arthroscopy 25(2):200–207

4. Bedi A, Kawamura S, Ying L, Rodeo SA (2009) Differences in

tendon graft healing between the intra-articular and extra-artic-

ular ends of a bone tunnel. HSS J 5(1):51–57

5. Brady MF, Bradley MP, Fleming BC, Fadale PD, Hulstyn MJ,

Banerjee R (2007) Effects of initial graft tension on the tibio-

femoral compressive forces and joint position after anterior cru-

ciate ligament reconstruction. Am J Sports Med 35(3):395–403

6. Guillard C, Lintz F, Odri GA, Vogeli D, Colin F, Collon S,

Chappard D, Gouin F, Robert H (2012) Effects of graft preten-

sioning in anterior cruciate ligament Reconstruction. Knee Surg

Sports Traumatol Arthrosc. doi:10.1007/s00167-011-1833-1

7. Kim SG, Kurosawa H, Sakuraba K, Ikeda H, Takazawa S (2006)

The effect of initial graft tension on postoperative clinical out-

come in anterior cruciate ligament reconstruction with semiten-

dinosus tendon. Arch Orthop Trauma Surg 126(4):260–264

8. Labs K, Perka C, Schneider F (2002) The biological and bio-

mechanical effect ofdifferent graft tensioning in anterior cruciate

ligament reconstruction: an experimental study. Arch Orthop

Trauma Surg 122(4):193–199

9. Mae T, Shino K, Matsumoto N, Natsu-Ume T, Yoneda K,

Yoshikawa H, Yoneda M (2010) Anatomic double-bundle ante-

rior cruciate ligament reconstruction using hamstring tendons

with minimally required initial tension. Arthroscopy 26(10):

1289–1295

10. Mae T, Shino K, Nakata K, Toritsuka Y, Otsubo H, Fujie H

(2008) Optimization of graft ﬁxation at the time of anterior

cruciate ligament reconstruction. Part I: effect of initial tension.

Am J Sports Med 36(6):1087–1093

11. Nicholas SJ, D’Amato MJ, Mullaney MJ, Tyler TF, Kolstad K,

McHugh MP (2004) A prospectively randomized double-blind

study on the effect of initial graft tension on knee stability after

anterior cruciate ligament reconstruction. Am J Sports Med

32(8):1881–1886

12. Pen˜a E, Martı´nez MA, Calvo B, Palanca D, Doblare´ M (2005) A

ﬁnite element simulation of the effect of graft stiffness and graft

tensioning in ACL reconstruction. Clin Biomech (Bristol, Avon)

20(6):636–644

13. Reese SP, Maas SA, Weiss JA (2010) Micromechanical models

of helical superstructures in ligament and tendon ﬁbers predict

large Poisson’s ratios. J Biomech 43(7):1394–1400

Knee Surg Sports Traumatol Arthrosc

123

Appendix IIIA

Adding text to pdf file - insert text into PDF content in C#.net, ASP.NET, MVC, Ajax, WinForms, WPF

XDoc.PDF for .NET, providing C# demo code for inserting text to PDF file

adding text to a pdf in reader; add text box to pdf

Adding text to pdf file - VB.NET PDF insert text library: insert text into PDF content in vb.net, ASP.NET, MVC, Ajax, WinForms, WPF

Providing Demo Code for Adding and Inserting Text to PDF File Page in VB.NET Program

how to insert pdf into email text; how to insert text in pdf reader

14. Ruth EB (1935) Metamorphosis of the pubic symphysis. I. The

white rat (Mus norvegicus albinus). Anat Rec 64:1–7

15. Wen CY,QinL, Lee KM, Chan KM (2009)Peri-graft bone mass

and connectivity as predictors for the strength of tendon-to-bone

attachment after anterior cruciate ligament reconstruction. Bone

45:545–552

16. Woo SL-Y, Hollis JM, Adams DJ, Lyon RM, Takai S (1991)

Tensile properties of the humanfemur-anterior cruciate ligament-

tibia complex: the effects of specimenage and orientation. Am J

Sports Med 19(3):217–225

17. Yoshiya S, Andrish JT, Manley MT, Bauer TW (1987) Graft

tension in anterior cruciate ligament reconstruction. An in vivo

study in dogs. Am J Sports Med 15(5):464–470

Knee Surg Sports Traumatol Arthrosc

123

Appendix IIIA

VB.NET PDF Page Insert Library: insert pages into PDF file in vb.

If you want to read the tutorial of PDF page adding in C# class, we suggest you go to C# Imaging - how to insert a new empty page to PDF file.

how to add a text box in a pdf file; add text to pdf acrobat

VB.NET PDF Library SDK to view, edit, convert, process PDF file

Feel free to define text or images on PDF document and extract accordingly. Capable of adding PDF file navigation features to your VB.NET program.

adding text field to pdf; add text pdf reader

Limb Idleness Index (LII): a novel measurement of pain in a rat model

of osteoarthritis

S.C. Fuyz**, Y.C. Cheukyz, L.K. Hungyz, K.M. Chanyz*

yDepartment ofOrthopaedicsand Traumatology, Facultyof Medicine, The Chinese UniversityofHong Kong, Hong Kong Special Administrative Region

zThe Hong Kong Jockey ClubSportsMedicine and HealthSciencesCentre, FacultyofMedicine, The Chinese University of Hong Kong, Hong Kong Special Administrative Region

a r t i c l e i n f o

Article history:

Received 27 February 2012

Accepted 2 August 2012

Keywords:

Osteoarthritis

Pain

Gait

Cartilage

Rat

s u m m a r y

Objectives: Mechanicalallodynia during ambulation inosteoarthritis (OA)animal models can be assessed

as decreased extentof loading or decreasedduration of loading. We propose to measure gaitadaptation

topain byboth mechanisms with the developmentof Limb Idleness Index (LII) in aratmodel of kneeOA.

Methods: Rats were assigned to anterior cruciate ligament transection (ACLT), Sham, or Normal group

(n ¼ 6). Gait data were collected at pre-injury, 1, 2, 3 and 6 months post-injury. Ratios of target print

intensity, anchor printintensity, andswing duration werecombinedtoobtain LII. The association of gait

changes withpainwasassessed by buprenorphine treatmentat3and6monthspost-injury. At6months,

OA-related structural changes in knee joints were examined by mCT and results fromhistological scoring

were correlated with LII.

Results: As compared to pre-injury level (range 0.75e1.20), LII in ACLT group was increased at 6months

post-injury, which was signiﬁcantly higher than that in Sham and Normal groups (P ¼ 0.024). The

increase in LII in ACLT group was effectively reversed by buprenorphine treatment (P ¼ 0.004). ACLT

group exhibited a signiﬁcantly higher maximum Osteoarthritis Research Society International (OARSI)

scoreas comparedtoSham (P¼ 0.005)and Normal(P ¼0.006)groups. Signiﬁcantcorrelationwas found

between LII and side-to-side difference in OARSI score (r ¼ 0.893, P < 0.001).

Conclusions: LII presents a good measurement for OA-related knee pain in rat model.

Introduction

Osteoarthritis (OA) is a debilitating progressive disease man-

ifested as pain in joints. The etiology and pathogenesis of OA is

not completely understood. Animal models for investigations of

OA are established to replicate the pathological changes

observed in clinical cases by genetic modiﬁcation, intra-articular

injections of chemicals such as mono-iodoacetate or surgical

destabilization of the joint

1,2

;yet there is no one gold standard

model for OA that sufﬁciently represent human etiology. OA

models induced by transection of anterior cruciate ligament

(ACLT) or menisectomy may represent a good mimic of post-

traumatic OA in humans, in spite of a faster disease progres-

sion

.In order to investigate new therapeutic strategies for OA,

we need to assess both symptom-modifying and disease-

modifying effects in these animal models.

Apart from histological and biochemical assessment, measure-

ment of pain is regarded as one of the major outcome measures,

which are available in several animal models,

in which half of

them OA changes are chemically induced

4e6

. These methods

successfully utilize paw elevation time

or weight-bearing

5,6

injured limbs to measure OA pain based on avoidance of pain-

triggering activities on the affected limbs (mechanical allodynia).

Because the avoidance mechanisms of mechanical allodynia could

be achieved by either decreasing duration of loading (increased

paw elevation time) or re-distribution of loading (decreased

weight-bearing), it is advisable to assess both mechanisms simul-

taneously.We propose to measure limb idleness by combining the

information of swing time of target limb (increased paw elevation

Part of the content in this paper has been presented (Paper#54) in the 58th

Annual Meeting of the Orthopaedic Research Society, 4e7th Feb, 2012, San

Francisco, CA, USA.

Address correspondence and reprint requests to: K.M. Chan, Department of

Orthopaedics and Traumatology, The Chinese University of Hong Kong, Prince of

Wales Hospital, Shatin, NewTerritories,Hong KongSpecialAdministrative Region.

Tel: 852-26322728; Fax: 852-26377889.

** Address correspondence and reprint requests to: S.C. Fu, Department of

Orthopaedics and Traumatology, The Chinese University of Hong Kong, Prince of

Wales Hospital, Shatin, New Territories, Hong Kong. Tel: 852-26323311; Fax: 852-

26377889.

E-mail addresses: : bruma@cuhk.edu.hk (S.C. Fu), kaimingchan@cuhk.edu.hk

(K.M. Chan).

1063-4584/$ esee front matter 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. Allrights reserved.

http://dx.doi.org/10.1016/j.joca.2012.08.006

OsteoarthritisandCartilage xxx(2012) 1e8

Please cite this article in press as: Fu SC, et al., Limb Idleness Index (LII): a novel measurement of pain in a rat model of osteoarthritis,

Osteoarthritis and Cartilage (2012), http://dx.doi.org/10.1016/j.joca.2012.08.006

Appendix IIIB

VB.NET PDF Text Box Edit Library: add, delete, update PDF text box

code below to your VB.NET class program for adding text box on Dim outputFilePath As String = Program.RootPath + "\\" Annot_9.pdf" ' open a PDF file Dim doc

how to insert text box in pdf document; adding text to pdf document

C# PDF Text Box Edit Library: add, delete, update PDF text box in

for adding text box to PDF document in .NET WinForms application. A web based PDF annotation application able to add text box comments to adobe PDF file online

acrobat add text to pdf; adding text to pdf in reader

time to avoid loading), paw intensity on target limb (decreased

weight-bearing to avoidloading) andpaw intensity onanchor limb

(increased sharing of weight from target limb), using a Catwalk

animal gait analysis system. Because walking speed will affect all

gait parameters and the rats are allowed to walk freely without

constraint on speed, internal control for run-to-run variations in

walking speed can be achieved by normalization to contralateral

side. A Limb Idleness Index (LII) is calculated as a product of the

ratios of target paw print intensity,anchor paw print intensity and

swing duration. We hypothesize that LII is useful to detect limb

idlenessrelatedto OA pain.Inthepresentstudy,wemeasuredLII in

arat model of knee OA induced by ACLT and determined its rela-

tionship to pain by analgesic reversal test. Moreover, whether the

pain-related limb idleness was contributed by OA changes was

evaluated by histological examination and micro-computed

tomography (mCT).

Methods

Rat OA model induced by ACL transection

The animal experiments were approved by the Animal Ethics

andExperimentationCommitteeof TheChineseUniversityofHong

Kong(Reference No:11/008/DRG) andall animalsreceivedhumane

care.Eighteen female SpragueeDawley rats,12 weeks old, with an

averagebody weight of220 g,wereusedinthe present study. The

rats were randomly assigned to Normal, Sham and ACLT groups

(n ¼ 6). In the ACLT group, a well-established protocol of ACL

transectionwasadaptedto inducekneeOA

7,8

.Inbrief,theratswere

anaesthetized by intra-peritoneal injection of ketamine and xyla-

zine (75 and 10 mg/kg body weight respectively) A medial para-

patellar arthrotomy was made, followed by a lateral displacement

of patella and full ﬂexion of the knee to expose the intra-articular

joint space. The ACL was then transected with micro-scissors and

successful ACL transection was assured by a positive anterior

drawer test. After rinsing the joint space with saline, the joint

capsuleandtheskinwereclosedinlayers.IntheSham group,sham

operation was performed with the same procedures except ACL

transection. The rats were allowed free cage movement immedi-

ately after surgery. In the Normal group, no operation was

performed.

Catwalk animal gait analysis

Animal gait analysis was performed by Catwalk XT 9.0 (Nol-

dus Information Technology, Wageningen, The Netherlands) at

pre-injury, 1, 2, 3 and 6 months post-injury. The animals were

put on the walkway to familiarize with the settings 1e2 days

before the day of assessment. The camera was set at 60 cm

from the walkway and the region of interest (ROI) (8  20 cm)

for image capture was deﬁned and calibrated. All rats were

weighed before gait analysis, and the rat with a body weight

closest to the average value was used to set thresholds to pick

up the illuminated contact prints [Fig. 1(A)]. The rats were

allowed to walk voluntarily back and forthinside the walkway in

the dark; video recording of paw prints [Fig. 1(B)] was auto-

matically triggered when the rats entered the ROI.Recorded runs

[Fig. 1(C)] with a steady walking speed (variation <30%) were

accepted as compliant runs for paw print auto-classiﬁcation as

left front (LF), right front (RF), left hind (LH) and right hind (RH)

by the built-in software and the correctness of classiﬁcation

was further checked manually. Stance duration was detected as

the time of paw contact [colored bar in n Fig.1(D)] and swing

duration was detected as the time between consecutive paw

contacts. Only runs with normal alternate footfall pattern

(LF 0 RH 0 RF 0 LH) [Fig. 1(E)] were included for further

analysis. Three to ﬁve runs were kept for calculation of gait

parameters for every trial.

Calculation of LII

Gait parameters were presented as ratios between target

(injured) side and contralateral side to control for run-to-run and

individual variations. The extent of limb loading during walking

was evaluated by the paw print intensities. In the present study

with injured RH in ACLT and Sham groups, if RH was idled during

walking, the loading on target limb (RH) might be decreased with

adimmer or even smaller paw print; alternatively, the loading on

theanchor limb (LF)might be increasedto sharetheloadingon RH.

Thus the target print ratio (LH/RH) and the anchor print ratio (LF/

RF) were increased in case of limb idleness on RH. Because a paw

print is composedof a series of contact prints during stance phase

[Fig.1(F)], paw print intensity was calculated as the integration of

Fig.1. Limbloadingduringwalkingisevaluatedbycapturingilluminated pawprintsbyCatwalksystem(A).The pawprintsare automaticallyclassiﬁedafterimagecapture (B).The

resultingpaw printpatterns(C)areusedtomeasure parameterssuch asprintarea,printintensityand swingdurationforall four limbs.Arunwithsteadyspeedhaslittle variations

in stance and swingdurationsasshownbythe lengthsofcolor bar in(D),which isqualiﬁedfor further analysis.Normallyratswalkinan alternate footfallpattern(E)andonlythe

runswiththisfootfallpatternareincludedforcalculation ofLII.Apawprint iscomposed ofa seriesofcontactprints(F)duringstance phase.Pawprintintensityiscalculatedasthe

integration ofcontactprint intensitiesfor all capturedtime framesduringstance phase.Contactprint intensityfor everytime frame iscalculatedbymultiplyingthemeanintensity

duringcontactwith the contactprint area(asshownin F).Normalized pawprintintensity(G) iscalculatedbydividingthe paw printintensitywith pawprintarea (asshowninB),

which is then used to calculate the target print ratioand anchor print ratiofor consecutive pairsof front pawsand hind pawsrespectively.

S.C. Fuetal. /Osteoarthritis and Cartilage xxx (2012) 1e8

Please cite this article in press as: Fu SC, et al., Limb Idleness Index (LII): a novel measurement of pain in a rat model of osteoarthritis,

Osteoarthritis and Cartilage (2012), http://dx.doi.org/10.1016/j.joca.2012.08.006

Appendix IIIB

C# PDF Annotate Library: Draw, edit PDF annotation, markups in C#.

Provide users with examples for adding text box to PDF and edit font size and color in text box field in C#.NET program. C#.NET: Draw Markups on PDF File.

add text box to pdf file; adding text to pdf reader

C# PDF Page Insert Library: insert pages into PDF file in C#.net

By using reliable APIs, C# programmers are capable of adding and inserting (empty) PDF page or pages from various file formats, such as PDF, Tiff, Word, Excel

add text to pdf file reader; how to insert text into a pdf using reader

contact print intensities for all captured time frames during stance

phase. Contact print intensity for every time frame was calculated

by multiplying the mean intensity during contact with the contact

print area (from the raw data output of the Catwalk software). The

paw print intensity was normalized with paw print area to take

accountfor thevariationsinthesizedifferencesintheleftandright

paws. The normalized paw print intensity was then used to

calculate target print ratio and anchor print ratio for consecutive

pairs of front paws and hind paws respectively [Fig.1(G)]. Apart

from decreased loading during stance phase, limb idleness during

walking could be achievedby increasing the swing duration. Thus

the swing duration ratio (RH/LH) also reﬂects limb idleness on

targetlimb.Becausetargetprint ratio, anchor print ratio andswing

duration ratio are three inter-dependent parameters representing

different aspects of gait adaptation for an idled limb, a product of

these three ratios will better reﬂect the extent of limb idleness

regardless of the idling mechanisms, and we call this quantity LII.

Wehavedevelopedthecalculation ofLII withapilot studyonACLT

andsham-operatedrats (datanot shown). The result ofthepresent

studywas avalidation oftheuseofLII to measurepain-related gait

changes in arat OA model as comparedto sham operation and un-

traumatized normal rats.

Reversal test by buprenorphine injection

After collection of gait data at 3 months and 6 months post-

injury, reversal test by a single intra-peritoneal injection of

buprenorphine (0.025 mg/kg body weight) was performed

.Gait

analysiswas performedagain at0.5 and24 h after buprenorphrine

administration.

mCT analysis

After the last session of gait analysis, the rats were euthanized

by intra-peritoneal injection ofoverdose pentobarbital (20% w/v).

Theharvestedkneejointsegments werescannedwithmCT (VivaCT

40, Scanco Medical AG,Brüttisellen,Switzerland) at aresolution of

35 mm. Analysis was performed by built-in software with opti-

mized Gaussianﬁlter (sigma: 0, support: 2) andthreshold(>255).

In brief, rectangular volume ofinterest inthe dimensions of 2 mm

(L)  1 mm (W) 0.5 mm (D) was put in the subchondral bone at

medial and lateral tibial epiphysis

.Trabecular indices including

bone volume/ tissue volume (BV/TV), connectivity density,

trabecular thickness,trabecular number andtrabecular separation

were determined. The knee samples were then ﬁxed in 10%

buffered formalin solution overnight for subsequent histological

processing.

Histological examination

The formalin-ﬁxed knee samples were decalciﬁed in 9% formic

acid for 2 weeks. Frontal sectioning was performed to obtain six

5mm sections at approximately 200 mm steps

.Hematoxylin and

Eosin staining was performed for scoring of osteoarthritic changes

according to the cartilage OA histopathology grading system from

OsteoarthritisResearchSociety International (OARSI)

.Inbrief,the

grade (0e6) and stage (0e4) of OA on the medial and lateral

articular compartments (revealing both femoral and tibial surface

in frontal sections) were scored by two-independent investigators

in a single-blinded fashion. A semi-quantitative OARSI score was

calculated by multiplying OA grade and OA stage, and the

maximum OARSI score among four ROIs (medial/lateral side of

femur/tibia) in the articular compartments in sampled frontal

sectionswas consideredas the most pathological regions for group

comparisons. Toluidine blue staining was also performed to

facilitate the examination of cartilage loss as one of the criteria of

OARSI score.

Statistical analysis

Statistical analysis was done using Statistical Package for Social

Science (SPSS) 16.0 (SPSS Inc, Chicago,USA).Because the ratio data

follow alognormaldistribution,gait datawerelog-transformedfor

parametric tests. The coefﬁcient of variation (CV) for LII measure-

mentwascalculatedfromtheaverage ofthevariancesofeachsetof

repeatedmeasurement(V),accordingto theequationCV ¼(e

1)

1/2

for log-transformed data. Repeated measure Analysis of Variance

(ANOVA) was used to analyze the log-transformed gait data with

respect to temporal changes/buprenorphine treatment (within-

subject factor) and experimental groups (between-subject factor).

Comparisons ofgait databeforeandafter buprenorphinetreatment

in each experimental group were performed by paired t test if the

within-subjecteffectwasstatisticallysigniﬁcant.For comparisonsof

end-point measurement at 6 months post-injury, mCT data were

analyzed by one-way ANOVA with post-hoc Turkey’s test after

checking for data normal distribution; while gait data and OARSI

scores were analyzed by non-parametric KruskaleWallis test, and

post-hoc two-group comparison by ManneWhitney test with

Bonferroni correction if necessary. Correlation between LII and

OARSI scores was performed by Spearman’s rho test. Signiﬁcant

difference was determined at P < 0.05.

Results

Among the 18 rats,1 rat from Sham group died at 1 week post-

injury due to post-surgical infection. Gait data for this rat were

collected at pre-injury only.

Gait changes in ACLT rat model

The average of the variances of each set of repeated measure-

ment for log-transformed LII was 0.018. The CV for LII was calcu-

latedas 13.3%,indicatingacceptablereliabilityofthemeasurement.

At pre-injury time point (n ¼ 18), the mean LII was 0.97 with

a range of 0.75e1.20. Although the between-subject effect

(different experimental groups) on LII was not statistically signiﬁ-

cant (repeated measure ANOVA with between-subject effects,

P ¼ 0.273), the within-subject effects of time post-injury

(P ¼ 0.042) and time/group interaction (P ¼ 0.024) was signiﬁ-

cant, indicating the temporal changes of LII were different among

the experimental groups. Target print ratio, anchor print ratio and

swing duration ratio did not show signiﬁcant differences with

respect to within-subject effects (P ¼ 0.100, 0.816, 0.051, respec-

tively) and between-subject effects (P ¼ 0.213, 0.397, 0.201,

respectively).Although there wereobvious individual variations in

the gait data, LII in ACLT group was signiﬁcantly increased

(Repeated measure ANOVA within-subject effect only, P ¼ 0.024)

until 6 monthspost-operation(Fig.2),withall ACLT rats gothigher

LII as comparedto pre-injurylevels. One rat (rat54) inSham group

and two rats in Normal group (rat62, 63) also exhibited increased

LII, but the temporal gait changes in Sham and Normal groupwere

not signiﬁcant (P ¼ 0.214, 0.147, respectively). In ACLT group, the

signiﬁcanttemporal gaitchangeinLII wascontributedmainly byan

increased target print ratio (P ¼ 0.008) and swing duration ratio

(P ¼ 0.010), but not by anchor print ratio (P ¼ 0.180). When

comparisons were made at 6 months post-operation by Kruskale

Wallis test, ACLT group exhibited a higher LII as compared to Sham

and Normal groups (P ¼ 0.029), while no signiﬁcant difference was

detected in target print ratio (P ¼ 0.081), anchor print ratio

(P ¼ 0.134) andswing duration ratio (P ¼ 0.066).

S.C. Fuet al. /Osteoarthritis and Cartilage xxx (2012) 1e8

Please cite this article in press as: Fu SC, et al., Limb Idleness Index (LII): a novel measurement of pain in a rat model of osteoarthritis,

Osteoarthritis and Cartilage (2012), http://dx.doi.org/10.1016/j.joca.2012.08.006

Appendix IIIB

VB.NET PDF Text Add Library: add, delete, edit PDF text in vb.net

convert PDF to text, C#.NET convert PDF to images, C#.NET PDF file & pages Professional VB.NET Solution for Adding Text Annotation to PDF Page in VB

adding text fields to a pdf; how to add text to a pdf in preview

C# PDF File & Page Process Library SDK for C#.net, ASP.NET, MVC

VB.NET read PDF, VB.NET convert PDF to text, VB.NET an (empty) page to a PDF and adding empty pages Certainly, random pages can be deleted from PDF file as well

how to add text field to pdf; add text block to pdf

Detection of pain-related gait changes in response to analgesic

treatment

At 3 months post-injury, buprenorphine treatment did not

signiﬁcantly alter LII in all experimental groups (within-subject

effect P ¼ 0.922). At 6 months post-injury, buprenorphine treat-

ment effectively decreased LII (within-subject effect P ¼ 0.004) in

ACLT groupat 0.5 h after injection(paired ttest,P ¼ 0.041), andLII

wasincreasedat 24 hafter injection (P ¼0.014).Sham andNormal

groups did not respond to buprenorphine treatment (within-

subject,P ¼0.750,0.555, respectively).The responses of individual

rats to buprenorphine treatment with respect to different gait

parameters were showninFig.3. The LII ofall ACLT ratsresponded

to buprenorphine treatment, but target print ratio, anchor print

ratio and swing duration ratio failed to reveal the analgesic

response in some ACLT rats. The rats with high LII in Sham group

(rat54) and Normal group (rat62,63) also responded to buprenor-

phine treatment.

mCTanalysis of subchondral bone changes and correlation with LII

In mCT measurements on tibial subchondral bone, BV/TV

(Turkey’stest,P ¼0.026)andtrabecular thickness(P ¼0.006)inthe

lateral compartment was signiﬁcantly lower in ACLT group as

compared to Normal group, while the differences between Sham

and ACLT was not statistically signiﬁcant in these parameters

(P ¼ 0.406, 0.079, respectively). In the medial compartment, the

trabecular thickness (P ¼ 0.018) was also lower in ACLT group as

compared to Normal group, but connectivity density was signiﬁ-

cantly higher (P ¼0.012). The differences betweenSham and ACLT

were not statistically signiﬁcant in these parameters (P ¼ 0.167,

0.085, respectively). There was no signiﬁcant difference between

different groups withrespect to trabecular number and trabecular

separation (TableI).

Histological scoring for OA changes and correlation with LII

Histological scoring based on OARSI system showed that ACLT

group exhibiteda signiﬁcantly higher maximum score among four

ROIs (medial/lateral side of femur/tibia) on the target side, as

compared to both Sham (ManneWhitney test, P ¼ 0.005) and

Normal (P ¼ 0.006) group (TableI); while the difference of OARSI

scores on the contralateral side was not signiﬁcant (ACLT vs Sham

P ¼ 0.260; ACLT vs Normal P ¼ 0.042) (Bonferroni correction of

ManneWhitney U test for three comparisons, statistical signiﬁ-

cancewasacceptedatP<0.0167).Themostpathologicalsiteinthe

target limb in ACLT group was the medial femur. It was character-

ized by complete erosion of hyaline cartilage with formation of

sclerotic bone that involved 25e50% of articular surface in the

observed sections [Fig.4(A)]. The contralateral side in ACLT group

Fig. 2. The temporal changesoftarget print ratio (A),anchor print ratio (B), swing duration ratio (C) and LII(D) for individual rats are shownwith reference lines indicatingthe

normal range of the pre-injury levels.

S.C. Fuetal. /Osteoarthritis and Cartilage xxx (2012) 1e8

Please cite this article in press as: Fu SC, et al., Limb Idleness Index (LII): a novel measurement of pain in a rat model of osteoarthritis,

Osteoarthritis and Cartilage (2012), http://dx.doi.org/10.1016/j.joca.2012.08.006

Appendix IIIB

also exhibited signiﬁcant osteoarthritic changes [Fig. 4(D)]. The

OARSI scoresinbothtargetandcontralateralsides(P¼0.825,0.112,

respectively) were not signiﬁcantly different betweenSham group

andNormal group.Sham groupshowedpathological changesinthe

lateral tibia in both target and contralateral sides; which was

characterized by mid zone excavationor abnormal cysts

in <10%

ofarticular surface[Fig.4(B andE)].InNormal group,osteoarthritic

change was detected in some rats. In rat64, mid zone excavation

was found in medial tibia in the target side [Fig. 4(C)], while

mild osteoarthritic change was noticed in the contralateral side

[Fig.4(F)]. The OARSI scores of target and contralateral sides of

individualratswereshowninFig.5(A).Therelationshipofbetween

LII and the side-to-side difference of OARSI scores (target to

contralateral sides) was examined in a scatter plot [Fig. 5(B)].

Signiﬁcant correlation was detected with (r ¼ 0.862, P < 0.001) or

without (Spearman’s rho r ¼ 0.893, P < 0.001) the outlier datum

fromrat48,inwhichtendoncalciﬁcationwasobservedinthetarget

side in addition to osteoarthritic changes [Fig.5(C)]. The contra-

lateral side of rat48 also got signiﬁcant osteoarthritic changes

without tendon calciﬁcation [Fig.5(D)].

Table I

Results ofmCTmeasurementsand OARSIscores at 6 monthspost-injury

Parameters

Side

Normal(n ¼6)

Sham (n ¼5)

ACLT(n¼ 6)

Pvalues

BV/TV

Medial

0.454 (0.398e0.509)

0.399 (0.345e0.454)

0.386(0.296e0.476)

0.201

Lateral

0.359 (0.302e0.416)

0.303 (0.247e0.359)

0.254(0.173e0.335)

0.033

Connectivitydensity(1/mm3)

Medial

23.4 (13.4e33.5)

30.2 (20.5e39.8)

48.3(29.4e67.2)

0.014

Lateral

40.4 (27.9e52.9)

36.3 (23.7e48.9)

52.7(30.1e75.2)

0.222

Trabecularnumber (1/mm)

Medial

8.44 (8.14e8.74)

8.89 (7.83e9.95)

8.48(7.93e9.03)

0.398

Lateral

9.05 (8.47e9.62)

8.55 (7.89e9.21)

8.48(7.90e9.06)

0.185

Trabecularthickness(mm)

Medial

0.128 (0.115e0.141)

0.120 (0.111e0.129)

0.105(0.090e0.121)

0.022

Lateral

0.104 (0.092e0.117)

0.096 (0.082e0.110)

0.079(0.066e0.092)

0.007

Trabecularseparation (mm)

Medial

0.149 (0.136e0.163)

0.155 (0.143e0.167)

0.152(0.139e0.1

0.709

Lateral

0.161 (0.145e0.176)

0.161 (0.148e0.173)

0.163(0.150e0.177)

0.929

Maximum OARSIscore

Target

3.5 (1e12)

4(0e4.5)

18(6e20)

0.005

Contra-lateral

1(0e4)

4(1e4.5)

4.5(0e9)

0.069

Data arepresented asmean (lower andupperboundsof 95% conﬁdence interval) for mCTdata andmedian (minimumtomaximum) for OARSIscore. Pvalues for one-way

ANOVAareshown for themCTmeasurement; whilePvaluesfor KruskaleWallistest areshown forOARSI scores.Bold valuessignify P< 0.05.

Fig.3. The target print ratio(A), anchor print ratio(B), swingdurationratio(C) and LII(D)for individual rats at 6monthspost-operationbefore buprenorphine injection, at 0.5h

and at 24 h after injection are shownwith reference linesindicating the normal range ofthe pre-injurylevels.

S.C. Fuet al. /Osteoarthritis and Cartilage xxx (2012) 1e8

Please cite this article in press as: Fu SC, et al., Limb Idleness Index (LII): a novel measurement of pain in a rat model of osteoarthritis,

Osteoarthritis and Cartilage (2012), http://dx.doi.org/10.1016/j.joca.2012.08.006

Appendix IIIB

Discussion

Our results showed that LII is useful to reveal OA-related pain.

The CV for LII is 13.3%, indicating acceptable reliability of the

measurement. LII is in essence a measure of symmetry inwalking

gait.LII >1 means that thetarget limb wasidled;in contrast,LII <1

reﬂects less activity in the contralateral limb. At pre-injury time

point (n ¼ 18),the mean LII was 0.97 with a range of 0.75e1.20. In

the reversal test with buprenorphine injection, all rats with LII

1.20 were responsive to analgesic treatment, indicating limb

idleness on the target sides was associated with pain. On the

contrary,one rat from Sham group(rat59) with LII 0.75 indicated

signiﬁcant limb idling on the contralateral side. Rat59 also

responded to buprenorphine in a reversed way as compared to

other painful cases in target limbs (Fig.3). Examination on histo-

logical samples from rat59 showed that the OARSI score in

contralateral side (4.5) was higher than the target side (1). It

indicates that rat59 might experience pain associated with

osteoarthritic changes onthecontralateralside.Theseobservations

suggest that LII is sensitive to detect the relative mechanical allo-

dyniaofbothlimbs.Thusitis necessaryto consider bothtargetand

contralateral sides whenwe examinetherelationshipofLII andOA

changes. The signiﬁcant correlation between LII and the side-to-

side difference of OARSI scores supports that LII is capable of

detecting the relative severity of OA changes in both knees in the

rat model of ACLT.

This is theﬁrst study measuring OA painina surgically induced

OA animal model with a long follow-up (6 months), while all

previous animal studies of OA pain were carriedout ina relatively

shorter duration (maximum follow-up ranging from 2 weeks

10 weeks

).In thosestudieswithshorter follow-uptime, paindue

to acute joint inﬂammation rather than chronic arthritic changes

wasmeasured.

Ontheother hand,ascomparedto previousstudies

on rat ACLT model of knee OA,

15,16

our data showed a slower

development of OA (symptomatic at 6 months post-operation),

whichmight bedue tothe use offemale rats (221.5 11.9 g at pre-

Fig.4. H&Estainingoffrontalsectionsofthe rat kneesfromACLT(A,D),Sham(B,E) andNormal(C,F)groups(representativesampleswithmedianmaxOARSIscoresonthetarget

side) were shown.In ACLTrat(rat52),severe osteoarthriticchangeswith denudation wasobserved in the operate side (A); while mildsuperﬁcial zone delamination wasfound in

the contralateral knee (D).In Shamrat(rat55),mild cavityformationina circumscribedcartilage volume (excavation) wasnoticed in the lateral tibia (B),andsimilarlesions were

alsoobserved inthecontralateral side(E).InNormalrat(rat64),excavationwasalsoobserved inmedial tibia(C),while mild osteoarthriticchange wasobservedinthe contralateral

side (F).The grade and stage of OARSIscoresare shown in the ﬁguresandthe pathological sites are marked by: (optical magniﬁcation: 50).

S.C. Fuetal. /Osteoarthritis and Cartilage xxx (2012) 1e8

Please cite this article in press as: Fu SC, et al., Limb Idleness Index (LII): a novel measurement of pain in a rat model of osteoarthritis,

Osteoarthritis and Cartilage (2012), http://dx.doi.org/10.1016/j.joca.2012.08.006

Appendix IIIB

injury and 286.4  23.0 g at 6 months post-operation), as heavier

andmoreactivemalerats(around300g at 8 weeksold)areusedin

previous studies

.Yet we also detected similar subchondral bone

changes

and cartilage degeneration

15,16

.With such alongfollow-

up time, some spontaneous OA

were observed in the un-

traumatized knees. The observed outcomes in ACLT group were

probably contributedby both post-traumatic and primary OA. The

signiﬁcant intra-group variations in our data may affect the

detection of between-subject group differences. However, these

variations helped to explore the relationship of the painful

responses as detected by gait adaptation and the osteoarthritic

changes as revealed by histology. The correlation of LII to OARSI

scores suggested that the gait adaptation to pain was associated

with the OA-speciﬁc joint degeneration, with an outlier (rat48)

which was also explainable by the co-existence of OA and tendon

calciﬁcation. As abnormal LII was responsive to buprenorphine

treatment, the use of LII in ACLT rat model may be helpful for the

development of pain medication for OA patients, together with

examination of OA changes by histology.

Gait analysis has been used to measure this movement-evoked

pain in OA using Catwalk system

.All the built-in gait parameters

did not show signiﬁcant association with OA pain except for the

percentageof ipsilateral paw intensity at standing,whichis similar

to the calculation ofanchor print ratio andtarget print ratio in this

study. We calculated mean integrated paw print intensity to esti-

mate theextent oflimb loadingduring ambulation,whichmightbe

more representative than maximum or mean print intensities at

any time point during the stance phase (built-in gait parameters).

As limb idleness can also result from decreasing time of loading

(increasing paw elevation time) in addition to reducing loading

during stance phase, LII presents a new parameter that integrates

both mechanisms to avoid mechanical allodynia. The three

component ratios of LII are chosen because they represent three

differentaspectsto idleatarget limb during walking,thusLIIwould

be able to detect “limb idleness” when one of these ratios was

increased. For example, rat53 would be regarded as “no change in

limb idleness”ifweaccessedanchor print ratio andswing duration

ratio; but with a higher target print ratio, we concluded that rat53

also experienced limb idleness on target limb at 6 months post-

injury as revealed by LII (Fig.2), which also responded to bupre-

norphine treatment (Fig. 3). Alternatively, false-positive cases of

“limb idleness” detected by single ratio could be excluded when

other ratios were changed in different directions. For example,

rat55 got a high target print ratio but a low swing duration ratio,

thus it would be difﬁcult to judge whether rat55 was idling its

target limb if we only based on single ratio. By combining these

component ratios to yield LII, rat55 was still within the normal

range and it did not respond to buprenorphine treatment (Fig.3).

This may explain why individual component ratios cannot detect

the group difference at 6 months post-injury in contrast to LII,

because the rats in one group may not use the same strategy to

achievelimb idleness.Nevertheless,individual ratios shouldstill be

Fig.5. Calciﬁcationofpatellartendonwasdetected intheoperatedsideofrat48(LII:3.64)bymCTimagingin additiontoosteoarthriticchangesasshowninhistological section(A),

while noectopiccalciﬁcationwasfoundinthe contralateral side butsigniﬁcantosteoarthriticchangewasalsoobserved (B).Ascatter plotbetweenLIIandside-to-sidedifferencein

maximumOARSIscore(C) suggesteda positive correlationbetweenpain-related gaitadaptationandosteoarthriticchanges.Reference linesare drawntoindicatethe normalrange

of the pre-injurylevels ofLII.MaximumOARSI scoresfor individual rats were shown in (D).

S.C. Fuet al. /Osteoarthritis and Cartilage xxx (2012) 1e8

Please cite this article in press as: Fu SC, et al., Limb Idleness Index (LII): a novel measurement of pain in a rat model of osteoarthritis,

Osteoarthritis and Cartilage (2012), http://dx.doi.org/10.1016/j.joca.2012.08.006

Appendix IIIB

presentedalong withLII inthegait patternanalysisto measureOA-

related pain development in the ACLT model.Ifthere is aswitch of

limb idling strategies or compensatory changes, evaluations on

individual ratios and LII will provide more information.

There are several limitations for the use of LII to measure

symptomatic OA in animal model. Firstly, it is only applicable for

unilateral injury. Any unanticipated changes on the contralateral

limb would have chance to confound the assessment of limb idle-

ness, such as spontaneous development of primary OA.

Buprenorphine-sensitivechanges inLII may mean changes inpain

levels in target side or contralateral side. Therefore, it is important

toassessthestatusofcontralateral sidewhenLII isusedtomeasure

painful responses in animal model of unilateral injury. Secondly,

although we detected signiﬁcant correlations between LII and

osteoarthritic structural changes, the mere co-existence of OA

changes and gait changes at one single time point might not

provide strong evidence for association. Longitudinal studies that

include data from more time points would be more appropriate.

Thirdly, LII only reveals activity-related pain,whilepaininOA may

have other elements which are not activity-related

.Thus LII may

reveal only parts ofthe whole picture for the development ofpain

medication in animal model. Finally, other functional changes

which are not related to pain may also affect limb idleness, for

example, knee stiffness, extension deﬁcit or altered limb coordi-

nationmay leadto changesin gait parameters andaffectsymmetry

in walking gait. Although LII cannot differentiate different painful

conditions (OA vs tendinopathies) that trigger limb idleness, it is

possible to extend the use of LII for evaluation of functional

recovery as a restoration of gait symmetry after unilateral injury,

such as tendon injuries, muscle weakness or some neural injuries.

Further exploration on the use of LII to monitor limb functional

recovery in animal models is possible.

Conclusion

LII isauseful parameterto measureOA-relatedkneepaininarat

model.

Author contributions

Experimental design and intellectual input: SCF, KMC,LKH.

Data acquisition, analysis, manuscript writing: SCF, YCC.

Final approval ofmanuscript: All authors.

Role of funding source

This study was supported by the Direct Grant (Reference No.:

2010.2.041) from The Chinese University of Hong Kong.

Conﬂicts of interest

All authors declare no conﬂicts of interest.

References

1. Little CB, Smith MM. Animal models of osteoarthritis. Curr

Rheumatol Rev 2008;4:175e82.

2. Little CB, Zaki S. What constitutes an “animal model of oste-

oarthritis” e the need for consensus? Osteoarthritis Cartilage

2012;20:261e7.

3. Smith M, Ghosh P. Experimental models of osteoarthritis. In:

Moskowitz R, Howell D, Altman R, Buckwalter J, Goldberg M,

Eds. Osteoarthritis. 3rd edn. Philadelphia: W.B. Saunders;

2001:171e99.

4. Tonussi CR, Ferreira SH. Rat knee-joint carrageenan incapaci-

tation test: an objective screen for central and peripheral

analgesics. Pain 1992;48:421e7.

5. Bove SE, Calcaterra SL, Brooker RM, Huber CM, Guzman RE,

Juneau PL, et al. Weight bearing as a measure of disease

progression and efﬁcacy of anti-inﬂammatory compounds in

a model of monosodium iodoacetate-induced osteoarthritis.

Osteoarthritis Cartilage 2003;11:821e30.

6. Ferreira-Gomes J, Adães S, Castro-Lopes JM. Assessment of

movement-evoked pain in osteoarthritis by the knee-bend

and CatWalk tests: a clinically relevant study. J Pain 2008;9:

945e54.

7. Stoop R, Buma P, van der Kraan PM, Hollander AP, Clark

Billinghurst R, Robin Poole A, et al. Differences in type II

collagen degradation betweenperipheral and central cartilage

of rat stiﬂe joints after cranial cruciate ligament transection.

Arthritis Rheum 2000;43:2121e31.

8. Castro RR, Cunha FQ, Silva Jr FS, Rocha FA. A quantitative

approachto measurejoint paininexperimental osteoarthritise

evidence of a role for nitric oxide. Osteoarthritis Cartilage

2006;14:769e76.

9. Fu SC, Chan KM, Chan LS, Fong DT, Lui PY. The use of motion

analysis to measure pain-related behaviour in a rat model of

degenerative tendon injuries. J Neurosci Methods 2009;179:

309e18.

10. Koh YH, Hong SH, Kang HS, Chung CY, Koo KH, Chung HW,

et al. The effects of bone turnover rate on subchondral

trabecular bone structure and cartilage damage in the osteo-

arthritis rat model. Rheumatol Int 2010;30:1165e71.

11. Gerwin N, Bendele AM, Glasson S, Carlson CS. The OARSI

histopathology initiative - recommendations for histological

assessments ofosteoarthritisin the rat.Osteoarthritis Cartilage

2010;18(Suppl 3):S24e34.

12. Pritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP,

Revell PA, et al. Osteoarthritis cartilage histopathology:

grading and staging. Osteoarthritis Cartilage 2006;14:13e29.

13. Roemhildt ML, BeynnonBD,Gardner-Morse M. Mineralization

of articular cartilage in the spragueedawley rat: character-

ization and mechanical analysis. Osteoarthritis Cartilage 2012,

http://dx.doi.org/10.1016/j.joca.2012.04.011.

14. Li N, Rivéra-Bermúdez MA, Zhang M, Tejada J, Glasson SS,

Collins-Racie LA,etal.LXR modulation blocksprostaglandin E2

production and matrix degradation in cartilage and alleviates

pain in a rat osteoarthritis model. Proc Natl Acad Sci USA

2010;107:3734e9.

15. Galois L, Etienne S, Grossin L, Watrin-Pinzano A, Cournil-

Henrionnet C, Loeuille D, et al. Dose-response relationship for

exercise on severity of experimental osteoarthritis in rats:

apilot study. Osteoarthritis Cartilage 2004;12:779e86.

16. Chou MC, Tsai PH, Huang GS, Lee HS, Lee CH, Lin MH, et al.

Correlation between the MR T2 value at 4.7 T and relative

water content in articular cartilage in experimental osteoar-

thritis induced by ACL transection. Osteoarthritis Cartilage

2009;17:441e7.

17. Pritzker K. Animal models for osteoarthritis: processes, prob-

lems and prospects. Ann Rheum Dis 1994;53:406e20.

18. Felson DT. Developments in the clinical understanding of

osteoarthritis. Arthritis Res Ther 2009;11:203.

S.C. Fuetal. /Osteoarthritis and Cartilage xxx (2012) 1e8

Please cite this article in press as: Fu SC, et al., Limb Idleness Index (LII): a novel measurement of pain in a rat model of osteoarthritis,

Osteoarthritis and Cartilage (2012), http://dx.doi.org/10.1016/j.joca.2012.08.006

Appendix IIIB

Documents you may be interested

mvc view to pdf itextsharp : Adding text to pdf file SDK control service wpf azure web page dnn 92015006322-part1593