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PhD confirmation

for Science Class^^

li gao

on 19 February 2014

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Transcript of PhD confirmation

Literaure review
Neuronal regulation
Response of bone to mechanical loading
By: Kelly Gao
supervisor: Dr Guo Xia

Department of Rehabilitation Sciences, HKPU
Let's check it out..
What is capsaicin-sensitive sensory neuron
Does capsaicin-sensitive sensory neuron contribute to the maintenance bone integrity?
An animal experiment in a rat functional disuse model

Funtional disuse model
Response of bone to mechanical loading
Disuse of Osteoporosis
Summary & research question
Disuse of osteoporosis (OP)
Sensitivity of bone to weightlessness
Management of disuse OP
Characterization of disuse OP
Summary and research question
Thank you..

OP is the most
common skeletal disease
characterized by
reduced bone strength
which results in
increased bone fragility
leading to
increase risk
of pathological
(NIH Consensus Development Panel,2001)

Increasd bone fragility & the pathological #

Low bone mass

Microarchitectural deterioration
Diagnosis of OP AND classification
Material & experimental design
32 female Sprague-Dawley rats
SPSS 20.0
Data: mean & SD
One-way ANOVA
- body weight among different groups
Pilot results & disscussion
Experiment II study
Main study
Feb - June 2014
July -Dec 2014
Jan-Apr 2015
May - Dec 2015
Data collection & analysis
Experiment II study
Animal experiment
Data collection of Experiment II
Data analysis &
Writing thesis
work plan
Nanoindentation test
Bone histomorphometry
Immunochemical staining
Mechanisms of unloading on bone tissue
Bone loading AND CGRP expression
Benefits of regular physical exercise on bone healthy
All animals were housed individually in a stainless steel cages in a temperature-controlled room
12:12 hour light: dark cycle
Standard rodent chew and water providing libitum
Body weight and over all health were carefully monitored

Experimental design
Capsaicin: (Sigma, St Louis, MO, USA)

at 3 time points & repeated every 2 weeks:
25 mg/kg 50 mg/kg 50 mg/kg

6 h 24 h

Groups: N=8
(C + HLS)
Week 5-8
Week 1,3,5,7
capsaicin injection
Week 5-8
Week 1,3,5,7
Vehicle injection
Sacrificed by carbon dioxide inhaling (Week 8)
Hindlimb Suspension
Capsaicin injection
Week 5- 8
Under anesthesia
An approximately 30 degree head-down tilt
About 2 cm above the cage bottom

Bone Histomorphometry
Nanoindentation test
Immunohistochemistry staining
Body weight

Rats had beeb sacrificed by CO2 inhaling
Tibia scanned with micro-CT and then embedded with PMMA
Tibia microarchitecture
Discussion current result
Summary of Literature review
There is no significant difference in weight among the groups
MAONVA p < 0.001
One-way ANOVA: BV/TV: p < 0.001
TB.N: p < 0.001
Tb.Th: p = 0.001
Tb.sp: p < 0.01
Ct A/TA p = 0.650
Dramatic bone loss in the trabeluar of epiphysis region
No change occured in the diaphysis

CGRP positive nerver fibers are abundant various site in the epiphysis

Offley 2005: 4 wks capsaicin, reduction in metaphyseal BMD, no change in diaphysis

The integrity of sensory innervation which closely related to trabecular volume & resembling closely to the HLS model in maintenance of bone microarchitecture
Multivariate ANOVA

Univariate test
- Each of the outcome measures
Bonferroni post hoc test
A significant level of 0.05
I would like to express my heartfet thanks to my supervisor Dr Guo Xia for guidance and kindly support

I would like to thanks to Prof. Qin' lab and Prof Shi's lab

Thanks also give to my fellow: Dr Zhang Zongkang, Mr Lau Yuenchi, Mr Yang Lin and Miss Xia Lu; Mr Kevin Po & Mr Omega,

Thanks for Dr.Raymond Chung for his statistical advice

Thanks and respect to all the animals

Deepest thanks to my family members
Statical analysis

A pyramidal diamond indenter tip is loaded at a given depth into the sample and load is held constant, leading to a creeping of the material below the tip

The sample will be under sectioning, polishing, cleaning and air-drying

Er & H using the Oliver- Pharr model (1992)
CGRP has anabolic effects on bone cells
CGRP & Osteoblast Osteoclast
Mechanisms of CGRP on bone tissue
Nervous system
Regulation &/ modulation of bone metabolism
Bone is innervated by nerve fibers
Bone cells connect to the nervous system through unmyelinated sensory neurons

Familiar dysautomia
Loss of unmyelinated axons
Neuropeptide signaling
Bone mineral density
Bone fragility

37 amino acid
from calcitionin gene -
spine terminals
peripheral endings

- synthesized in
unmyelinated sensory neurons

CGRP alpha & beta

Expression of
CGRP alpha
- participated in the
of bone
Physiological activation of CGRP is required the concurrent of TWO molecules

G-protein-coupled calcitonin receptor-like receptor (CRL)
Receptor activity-modifying protein (RAMPI)
Mechanism of Capsaicin-induced CGRP release
Calcium influx the TRPV1
Unmyelinated & some small diamteter myelinated sensory neurons

Destroyed unmylelinated & small diameter mylinated sensory neurons
Depletion of SP and CGRP in peripheral nerve

Motor, large sensory afferent & symapthetic fiber function Neuropeptide levels in the CNS

bone fracture healing
bone defect repair
bone metabolism & cells function

An efficient approach to examine the role of sensory system & would be able to build chemically selective sensory denervation animal experiments for bone investigation
Exposures to microgravity for astronauts
Paralysis or prolonged immobility for people with spinal cord injury (SCI)
Neurological disease
(Lau & Guo, 2011)

Decrease in bone mass
Microstructural deterioration

Risk of fracture

Exposures to microgravity
prolong exposure to microgravity
for exploration-class missions
(e. g. 6 months to space station at 2020 stated by China national space administration),
will leading to significant
loss of BMD
Bone strength
Sample et al., 2008

- Cyclic compression with mechanical load on diaphysis under anesthesia by bupivacaine

- Concentration of CGRP on loaded bone decreased

Sample et al., 2011
- Cyclic compression load on diaphysis induced fatigue + 10 days CGRP treatment

- CGRP increased
reparative bone formation

- 1.5mm below growth plate
- 1.0 mm above tibia-fibular junction up to a height of 0.6 mm

Bone volume fraction (BV/TV, %)
Trabecular number (Tb. N, 1/mm)
Trabecular thickness (Tb. Th, mm)
Trabecular separation (Tb. Sp, mm)
Cortical bone area fraction (Ct. A/TA, %)
Functional disuse in the rat hindlimb via the HLS procedure was modified from Morey-Holton & Globus (2002)
Summary of literaure review
1. To investigate the expression of neuropeptide CGRP, microarchitecture, nanomechanical property and cellular activity in bone under the influence of capsaicin and microgravity in a rat hindlimb suspension model.

2. To investigate the effects of CGRP on bone integrity using a functional disuse model.


1. Microgravity has effects on the microarchitecture, nanomechanical properties, cellular activity and CGRP expression of bone in a rat hindlimb suspension model

2. Capsaicin has effects on the microarchitecture, nanomechanical properties, cellular activity and CGRP expression of bone in a rat hindlimb suspension model

3. Both capsaicin and microgravity will have similar effects on bone integrity which reflects the role of CGRP in the contribution to bone health

4. CGRP has effects on the microarchitecture, nanomechanical properties, cellular activity and CGRP concentration of bone in a rat hindlimb suspension model

bone mass
Anabolic and repair effects
Effect of CGRP on unloading

WHO defined OP as the bone mineral density(BMD) 2.5 standard deviations below the mean for young female adult (WHO, 2004)

Primary type I
: post menopause women due to lack of endogeous estrogen

Primary type II
: age-related bone loss,both men & women

: effects of underlying disease / treatment of such disease, any age and gender
Pharmacological prevention & treatment

Disuse of OP:
Lack of effective treatment

Chan et al 2004
- RCT to evaluate the effects of TC on bone quality
- BMD in both TC & control, but slower rate in TC
- pQCT: 2.6- to 3.6 fold retardation of bone loss in distial tibia in TC
CGRP knockout model
- decreased bone formation rate
- accelerated bone loss with aging

- During osteoclastic bone resorption
- Inhibit osteoclastic resorption in vitro & in vivo

- Reparative new bone formation after load-induced bone fatigue
Representative 3D micro-CT images of the proximal tibia a) Control; b) Capsaicin;
c) Hindlimb suspension d) Combination
254 x254 (300x300 DPI)

The anabolic effect of CGRP on bone formation and resorption has been well documented

Previous studies have demonstrated the effects of CGRP on regulatory and reparative effect of load-induced microdamage in animal model.

Experiment I study: capsaicin-induced sensory denervation decrease CGRP expression in trabecular bone in the same manner with disuse osteoporosis by HLS

Experiment II What is the effect of CGRP on bone reparative after hindlimb suspension?
To investigate of the effects of CGRP in on bone integrity using a functional disuse model.

CGRP has effects on the microarchitecture, nanomechanical properties, cellular activity and CGRP concentration of bone in a rat hindlimb suspension model
Animals & HLS = experiment I
24 rats

CGRP immunostaining and fluorescence microscopy

- Innervation of CGRP-positive nerve fibers in the epiphysis of tibia
- Distribution

Decalcified with EDTA for 4 weeks
Embedded in OCT
Sectioned transversely into 12 micrometers thick

Dehydrated with isopropanol & embedded in PMMA

- 4 micrometers
- von Kossa & toluidine blue
- bone mass& architecture data with the light microscope
- 8 micrometers, unstain
- measurements of fluorochrome-based indices

Role of CGRP
in anabolic function on bone cells

1. Inhibit human OB apoptosis
2. Stimulating Wnt signaling
inhibiting of the enzyme
GSK-3 ß through
protein kinase A activation

Several mechanisms have been proposed on the
bone formation reduction using
rat hindlimb suspension model
1. Decreased numbers of OB

2. Reduction in normal mechanical loading of bone by muscle contraction

3. Osteocyte expression of sclerostin & of RANKL

Ren et al., 2012
- AP: longer duration & slower falling rate in DRG
- Conduction velocities decreased
- DRG sensory neuron more excitable

Ion currents changed?

Lab: bone morphological & expression of CGRP
2 weeks HLS with ES on the DRG
Wnt signaling play essential role in bone physiology

-Stablilization of

(Bloomfield et al., 2002; Wronski and Morey. 1983;
Wronski et al., 1987; Globus et al., 1984; Bikle and Halloran. 1999; Lang et al., 2006),
- Directly and accurately reflect the bone strength
- Resistance fracture

Whole bone strength
“the sum total of characteristics of the bone that influence the bone’s resistance to fracture”
- Bone mass
- Structural properties
- Materials properties

Traditional mechanical test
- combination of structural & materials level properties

(Valentijn et al.,1997; Schinke et al., 2004; Hara-Irie et al.,1996; Imai et al., 1997;Susannah et al, 2011)

- total tissue area
- bone area & bone perimeter,
- OC and OB surface,
- No. OC & OB

- single- and double-labeled perimeter and interlabel width
- bone volume,
- mineralizing surface
- mineral apposition rate
Calcitonin Gene -Related Peptides
Adapt to the changing of the bone structure
Bone marrow
trabecular bone
in bone cells
bone changes

(Rawadi and Roman- Roman, 2005; Chau et al., 2009)
Cell culture study on human OB
(Caternina et a;., 1997, 2001; Tominaga et al., 1998; Spitzer et al., 2008)
Capsaicin, neurotoxic agent
Capsaicin induced sensory denervation
(Adam et al., 2000; Ape; et al., 2009; Ding et al., 2010; Henmi et al., 2011)
Summary of neuronal regulation
Summary of loading
Summary of disuse OP
(Alexandre & Vico,2011; Lau & GUO, 2011)
Disuse refers bone loss & cause secondary OP due to mechanical unloading & leads an increase in resorption and a decrease in bone formation in adults

Effective treatment is lacking

(Bjurholm et al., 1998; Hukkanen et al., 1992; Mach et al., 2002).
(Offley et al., 2005)
Bjurholm A, Kreicbergs A, Brodin E, Schultzberg M. Substance P and CGRP – immunoreactive nerves in bone. Peptide 1988; 9:165-171.
Chen KM, Qin L, Lau MC, Woo J, Au, S, Choy WY, Lee KM, Lee SH. A randomized, prospective study of the effects of Tai Chi Chun exercise on bone mineral density in postmenopausal women.
Hay DL. What makes a CGRP2 receptor? Clin Exp Pharmacol Physiol 2007; 34:963-971.

Hukkanen M, Konttinen YT, Rees RG, Gibson SJ, Santavirta S, Polak JM. Innervation of bone from healthy and arthritic rats by substance P and calcitonin gene related containing sensory fibers. J Rheumatol 1992; 19: 1251-1259.
Lau RY, Guo X. A review on current osteoporosis research: with special focus on disuse bone loss. J Osteoporos 2011; 2011:293808.
Lerner UH, Persson E. Osteotropic effects by the neuropeptides calcitonin gene-related peptide, substance P and vasoactive intestinal peptide. J Musculoskelet Neuronal Interact 2008; 8:154-165.
Mach DB, Rogers SD, Sabino MC, Luger NM, Schwei MJ, Pomonis JD, et al. Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur. Neuroscience 2002; 113:155-166.

Offley SC, Guo TZ, Wei T, Clark JD, Vogel H, Lindsey DP, Jacobs CR, Yao W, Lane NE, Kingery WS. Capsaicin-sensitive sensory neurons contribute to the maintenance of trabecular bone integrity. J Bone Miner Res 2005. 20: 257-267.
Smith LJ, Schirer JP, Fazzalari P, Klaushofer K. The role of mineral content in determining the micromechanical properties of discrete trabecular bone remodeling packets. J Biomech 2010. 43:3144-3149.
Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR. Bone histomorphometry: Standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 1987; 2: 595-610.
Morey-Holton ER, Globus RK. Hindlimb unloading rodent model technical aspects. J Appl Physiol 2002. 92: 1367-1377.
NIH Consensue Development Panel. Osteoporosis prevention, diagnosis, and therapy. JAMA 2001; 285: 785-795.
Sample SJ, Behan M, Smith L, Oldenhoff WE, Markel MD…. Functional adapation to loading of a single bone is neuronally regulated and involves multiple bones. J Bone Miner Res 2008; 23:1372-1381.
Sample SJ, Hao ZL, Wilson AP, Muir P. Role of calcitonin gene-related peptide in bone repair after cyclic fatigue loading. Plos One 2011; 6: e20386.
Wronski TJ, Morey ER. Effect of spaceflight on periosteal bone formation in rats. Am J Physiol 1983; 244:R305-309
Wronski TJ, Morey-Holton, ER, Doty SB, Maese AC, Walsh CC. Histomorphometric analysis of rat skeleton following spaceflight. Am J Physiol 1987; 252: R252-R255.

- periosteum
- bone marrow
epiphyseal trabecular
- OB & OC receptors of CGRP
anabolic & repair effects

examine the role of sensory system

Astronauts from space missions of 6 months
- Loss of total bone mass 11% in proximal femur region
- Trabecular bone mass & density: 14.4 -16.5 %

Acute and chronic stage of SCI
- 20-43% reduction in BMD

HLS in animal
- Decrement in the mechanical properties
- Reduction in bone formation rates
- Decrease calcium content
- Lower BMD

Exposures to microgravity for astronauts, and paralysis (SCI) & neurological disease have lead to disuse osteoporosis

A decrease in bone mass and microstructural deterioration of the skeleton which leads to increased risk of fracture

pQCT & BMD cannot directly and accurately reflect the bone strength, or resistance fracture

Bone strength at material level could measure by nanoindentation test
4. Sensory innervation of bone could have potential effects on skeletal response to mechanical stimuli
DRG: Electrophysiological changes in HLS???
Chemical effect by anesthesia leading to sensory denervation

Response to mechanical loading resulting in changes in neuropeptide concentrations in loaded bone
CGRP after fatigue loading?
Neuronal signaling pathway which has regulatory effects on load-induced repair
responses within the skeleton

Neuronal signaling pathway which has regulatory effects on load-induced repair
responses to bone

The regulatory effect of CGRP on unloading is still questionable

Whether or not the innervation of bone has a functional role in the physiological responses of disuse bone
Questions of unloading?
Bone fragility
Whether or not the innervation of bone has a functional role in the physiological responses of disuse bone
Physiological response
Role of Neural regulation in the physiological responses of unloading
Animal models: (innervation of bone & disuse bone)
Capsaicin: Sensory denervation - depletion of CGRP
Hindlimb suspension: disuse osteoporosis - simulate exposure to microgravity

Ax of physiological response:
Bone morphology: Micro-CT
Biomechanical: Nanoindentation
Neuropeptide expression: CGRP expression
Cellular activity: bone histomorphmetry

What is the role of CGRP on treatment of disuse osteoporosis
Animal model of disuse osteoporosis:
Hindlimb suspension:disuse osteoporosis
CGRP treatment after hindlimb suspension
Ax of physiological response:
Bone morphology: Micro-CT
Biomechanical: Nanoindentation
Neuropeptide expression: CGRP expression & quantification
Cellular activity: bone histomorphometry

Microarchitecture; nanomechanical properties
Cellular activity; CGRP expression
(Lau & Guo, 2011) 27
Experiment 1
Experiment II
(Mark et al., 2011)
Primary OP
(10% tween-80, 10% ethanol and 80% saline)
Dempster et al 2012
Stimulatory effect on osteoblasts proliferation
Close contacts with osteoclasts
Modulated targeted remodeling of microdamage
Sensory nerve
fibers containing CGRP:
Trabecular bone continuously
alters its shape
- CGRP-containing fibers

- the remodeling of the bone structure
Disuse osteoporosis
Nanoindentation belongs to the new generation of hardness testing techniques to characterize biological materials such as cortical and trabeculae bone at microstructural level to obtain the biomechanical properties.
Osteomeasure software
4 Weeks HLS
10 D
10 D
(Hay, 2007; Lerner and Persson, 2008; Sample et al.,2011)
sacrificed 10 days after HLS
Ax: Experiment I &
Quantification of CGRP by ELISA
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