• Two methods used for analysis: • Manual: Merz Grid

• Two-way RM-ANOVA to assess region and diet effects on each parameter (p<0.05).

• Thank you to Carmel Fargason and Laura Kelly for their efforts in making this project a success.

Primate Femur Histomorphometry and Gene Expression: Effects of Chronic Alcohol Abuse on Bone

S. Frischhertz, D. Feng1, C. Les2, C. Pechey2, R.W. Siggins3, S. Nelson4, G.J. Bagby3,4, J. Dufour5, P.E. Molina3,4, M. Lopez1

1Laboratory for Equine and Comparative Orthopedic Research, School of Veterinary Medicine, Louisiana State University (LSU), Baton Rouge, LA, 2Bone and Joint Center, Henry Ford Health System, Detroit, MI, 3Department of Physiology, LSU Health Sciences Center, New Orleans, LA, 4Comprehensive Alcohol

Research Center, LSU Health Sciences Center, New Orleans, LA, 5Tulane National Primate Research Center, Covington, LA

• Problem: Alcohol abuse is a widely recognized health concern that negatively impacts many organ systems. • Chronic alcohol consumption leads to secondary osteoporosis with

decreases in bone formation, bone mass, and bone mineral density. • Specific mechanisms must be better understood to optimize therapeutic

intervention.

• Objective: This study was designed to relate the specific histomorphometric changes with osteoclastic and osteoblastic gene expression alterations in a primate model of alcohol abuse.

Materials • Seven 3-4 year old male rhesus macaques: 19 months of treatment via

indwelling intra-gastric tube • 3 primates received alcohol diet

• 13-14 kg/week (30% W/V in water) • 4 primates received isocaloric sucrose diet

• Proximal femora were harvested for analysis • Left femur: qRT-PCR mRNA analysis

• Isolated greater trochanter • Stored in RNAlater (Qiagen)

Bone Regions

Histomorphometry: Results

RT-qPCR: Methods and Results • 16 bone regions examined for each parameter: 945 individual measurements

This research was supported by grants 1T35AA021097, AA009803, and AA020312 from the National Institute of Alcohol Abuse and Alcoholism.

Introduction

Acknowledgements

0

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sucrosealcohol

• This study was limited by sample size, but the preliminary results suggest that disruption of bone homeostasis at the mRNA level by chronic alcohol exposure contributes to the specific histomorphometric alterations of secondary osteoporosis.

• While larger studies are warranted to further examine the effects of alcohol ingestion on bone remodeling, potential gene targets for treatment of patients suffering from the homeostatic bone alterations due to alcoholism are suggested by these study results. • Treatments reversing this ↓ osteoblastic and ↑ osteoclastic activity may benefit alcoholic

patients suffering from secondary osteoporosis. Monoclonal antibody binding RANKL (Denosumab) may reverse the effects of ↓ OPG and recombinant parathyroid hormone (teriparatide) may help to ↑ lost osteoblastic function (for high risk cases). Bisphophonates may also be effective in reducing osteoclastic activity.

Conclusions

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Osteoclast 630x

Histomorphometry: Methods

• Right femur: Histomorphometry • Embedded in PMMA • Coronal sections • Toluidine blue stain

• • Digital bone images (Leica DFC480)

• • Manual surface tracing • • Software length and

area calculations

• Semi-automated: ImagePro v.5.01

Grid point Surface trace

Non-resorptive

surface

Resorptive

surface

Cement line

interface

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Osteoblastic Gene Expression Osteoclastic Gene Expression

Runt related transcription factor 2

(CBFA1) ↓

Cathepsin K (CTSK) ↓

Osteocalcin (OCAL) ↓ Matrix Metalloproteinase 9 (MMP9) ↑

Osteopontin (OPN) ↓ Tartrate resistant acid phosphatase

(TRAP) ↑

Osteoprotegerin (OPG) ↓ Receptor activator of nuclear factor

kappa beta (RANK) ↓

Collagen 1 alpha 1 (COL1A1) ↓ RANK ligand (RANKL) (no change)

Alkaline phosphatase (ALP) ↓

•Trends associated with alcohol consumption: •↓ expression of osteoblastic genes •Altered expression of osteoclastic genes •Activation: overall ↑ •Migration: ↑ •Resorption: possible net ↑

• Total RNA isolated from the left greater trochanters

• Messenger RNA levels of osteoblastic and osteoclastic target genes quantified with RT-qPCR using SYBRGreen technology

0

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Sucrose

Alcohol

Fo

ld C

ha

ng

e (

2-ΔΔ

Ct )

• Bone Volume/Tissue Volume

• Little change • Possible ↓ in mineralized

portion of total tissue volume

• Total Surface Length/Bone Area

* Significant ↑ in

cancellous bone of the alcohol group using Merz grid data

• Resorptive Surface Length/ Bone Area

• Mixed results for osteoclastic activity

• Non-resorptive Surface Length/Bone Area

• ↑ areas without osteoclastic activity

• Cement Line Interface Length/Bone Area

• ↓ remodeling activity

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• 7 Cortical regions •Femoral Neck

•Inferior (A) •Proximal Greater Trochanter

•Proximal (B), Medial (C), Lateral (D)

•Distal Greater Trochanter •Lateral (E)

•Distal Portion of the Proximal Metaphysis

•Medial (F), Lateral (G)

• 9 Cancellous regions •Femoral Head

•Inferolateral (a), Superolateral (b) •Femoral Neck

•Inferomedial (c), Superolateral (d) •Proximal Greater Trochanter

•Proximal (e), Lateral (f), Mid/Medial (g)

•Distal Greater Trochanter •Medial (h), Lateral (i)

• Five Outcome Measures • Bone volume/tissue volume (BV/TV)

• Mineralized portion of total tissue volume

• Resorptive surface length/bone area • Osteoclastic activity

• Non-resorptive surface length/bone area • Lack of osteoclastic activity

• Total surface length/bone area • Mineralized bone surface area

• Cement line interface length/bone area • Remodeling activity

• Ocular grid over bone • Manual assessments • Calculations

400x

Merz Grid ImagePro

a b

c d e g

f

h i

A

B C

D

E F

G

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