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Crocin Enhanced Functional Recovery after Sciatic Nerve Crush Injury in Rats

E.Tamaddonfard, A. Abbas Farshid, E. Ahmadian, A. Hamidhoseyn

Iranian Journal : Article in press : 1-8

Presented byRujikan Chaisanam

Neuroscience Program, Khon Kaen University

Introduction & Rationale

Objective

Materials & Methods

Results & Discussions

Conclusions

Sharing Opinions

Contents

The peripheral nerve

Sciatic nerve

Tibial nerve

Common peronealnerve

http://www.medicinenet.com/script/main/art.asp?articlekey=121208

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Tumors

Herniated disk Accident

Causes

Wallerian degeneration

Tissue damage + Macrophages acumulation, 24 hr after injury

Destruction of axons 36-48 hr after injury

Recruitment of Macrophages, myelin disintegration 48-72 hr after injury

Macrophages and Schwann cells scavenge Degenerated myelin, and Schwann cells

further proliferate and form Bünger bands3-7 day after injury

Classification of nerve injuryby Seddon

7

Neurapraxia

Axonotmesis

Neurotmesis

Presenter

Presentation Notes

Seddon had divided peripheral nerve injury into 3 levels The first one is Neurapraxia: A low severity injury that typically ทิปปิคคลี่ leads to complete recovery. The structure of the nerve remains intact but electrical conduction down แดว the ดิ axon is interrupted. Secondly, / Axonotmesis:/ Disruption of the neuronal axon takes place/ but the myelin sheath is still intact. This kind of injury caused by a crush based เบ่ส ท์ injury. And If the neuronal tubules are maintained in place, / regeneration /and restoration/ of sensory or motor ability may return. Regeneration rate was depending on the severity of the injury, / regeneration may occur over the weeks to years. And the last, Neurotmesis: �Characterized ไรสท์ by/ not only loss of nerve conduction, / but also /damage to surrounding nerve trunk connective tissue. This type of injury, / complete transsection occurs, / and commonly a neuroma forms/ over the proximal stump of the nerve during regeneration process, preventing normal continued regeneration to occur.

Nerve Injury Classification

Sunderland Seddon Injury Recovery Potential

I NeuropraxiaIonic block;

possible segmental demyelinization

Full

IIAxon severed;

Endoneurial tube intact

Full

III Endoneurial tube torn Slow; incomplete

IV Only epineuriumintact

Neuroma-in-continuity

V Loss of Continuity None

VI Combination of above Unpredictable

Axonotmesis

Neurotmesis

Presenter

Presentation Notes

However, / later เลเทอร์ on/ Sunderland expanded แพน it into five degrees /as you can see here. Grade 1: Neuropraxia Grade 2: Axonotmesis Disrupted axon /with intact endoneurium; / Wallerian degeneration takes place after one to two weeks Grade 3: Neurotmesis with preservation of perineurium /but Endoneurium is disrupted Grade 4: Neurotmesis with preservation of epineurium Grade 5: Neurotmesis with complete transection of nerve trunk For Grade 4 to 5 Nerve grafting is required As You can see here that/ after nerve injury/ Clinically คลินิคคอลลี่ approach/ are varied from conservative treatment /to surgery depending on severity of the injury.

Ketamine (80 mg/kg) and Xylazine(10 mg/kg)Crush

Sciatic Nerve Crush Injury Model

Pressure 0.5-1 kg/mm2

Presenter

Presentation Notes

In order to search for /a novel strategies to enhance regeneration after injury. Many animal models /have been proposed /but the Sciatic nerve crush injury animal model /is the most commonly used. I will briefly explain แพลน about the method / that induced Sciatic nerve crush injury After Rats were anesthetized. Theดิ area above the left lower thigh was shaved and sterilized. A 2-centimeter incision was made over the lateral aspect of the hind limb, and muscles are separated in order to expose the sciatic nerve. The nerve was crushed at 0.5 cm/ proximal to its trifurcation point /using a small haemostatic forceps. The nerve was crushed for one minute/ with an estimated pressure of/ 0.5-1 kilo gram per square millimeter. The crushed zone was approximately 4 square millimeter.

http://www.procedureswithcare.org.uk/wp-content/uploads/2010/07/DSC_2824.jpg

Nerve Crush Injury

Axon degeneration

Sciatic Nerve Crush Injury

Microtubule disassembly

Axon swelling

Axon fragmentation

Removal of remnant

Wallerian degeneration at distal stump :Schwann cells (SC)& Macrophage

Ischemia

Cell damaged:damages cellular

proteins, DNA, and the plasma

membrane

Release of more free radicals

Reperfusion

Reintroduces O2

White blood cells

Release a host of inflammatory factors :

interleukins (IL-1 )

Free radicalformation

Reduction in oxygenAnd energy substrates

Caspase dependent pathway

Apoptosis

E.protease & phospholipase

Introduction

&Gardenia jasminoides fruits Crocus sativus stigmas

Crocin: a naturalcarotenoid chemical compound

(Lee et al .2005 ; Aung et.al.2007)http://en.wikipedia.org/wiki/Crocin

A potent antioxidant

Anticarcinogenic

Aphrodisiac

Antidepressant

Neuroprotection

Introduction

( Abe et .al )

Crocin

Crocetin

Crocin powder ;from Fluka Schweizerland

?Nerve injury Functional recovery

Objective

Crocin

Materials & Methods

54 Male Wistar rat250 - 270g ( N =54)

Foot Print

Foot Print

Biochemical assays MDA

Euthanized

Biochemical assays MDA ,Histopathological ( H&E )

(Time line)Day -1 Day 15 Day 7,14,21,28 Day30

Sham operationcrush injury

Day 0 Day 1

IP injection 10 consecutive

days Gr1-Gr9

Gr6: Crush + NSS

(N=6)

Gr7: Crush+Crocin

5 mg/kg (N=6)

Gr8: Crush+Crocin

20 mg/kg (N=6)

Gr9: Crush+Crocin

80 mg/kg (N=6)

Crush IP injection 10 consecutive

Gr1 :

Intact+NSS

(N=6)

Gr2: Intact+Crocin

5 mg/kg (N=6)

Gr3: Intact+Crocin20 mg/kg (N=6)

Gr4:

Intact+Crocin

80 mg/kg (N=6)

Intact IP injection 10 consecutive

Gr5:

Sham surgery+NSS

(N=6)

Sham IP injection 10 consecutive

1.Rats hind feet were soaked with water soluble blue ink

Foot Prints Test

60 cm

7.5 cm

2.Rats were immediately allowed to walk along a confined walkway

Foot Print

Foot Print

(Time line)

Day -1 Day 7 Day30

Operation:Sham/

crush injury

Day 0

Foot Print

Foot Print

Day 14 Day 28

Sciatic functional index(SFI)

(Bain et .al)

Print length factor (PLF)

Toe spread factor (TSF)

Intermediary toe spread factor (ITS)

Undergoing sciatic nerve crush(E)

Normal limbs (N)

Sciatic functional index

0 = Normal function-100= Complete dysfunction

Lt Rt

PL

PL

TSTS

ITSITS

Malondialdehyde (MDA)

MDA, an index of free radical generation/lipid peroxidation

CH2(CHO)2

Ohkawa et al.1979

Sciatic Nerve

Histopathological evaluation

Fixed with 10% buffer formal saline

Dehydrated

Paraffin embedding serial longitudinal sectioning (4-5 μm)

Hematoxylin and Eosin (HE) staining

Statistical analysis

Mean ± SEM Two-way analysis of variance (ANOVA)

followed by Duncan’s test One-way ANOVA followed by Duncan’s test P< 0.05

Hypothesis

MDA levels

Histological changes in sciatic nerve: Swelling of Myelin sheet,Vacuolization and Myelin ellipsoids

Sciatic nerve crush injury

SFI Improved motor function

Wallerian degenerationDecreased free radicals

Crocin

A potent antioxidantNeuroprotection

Results & Disscussion

Result SFI

6 78 9

P< 0.05 represents significant difference vs crush + normal saline group

6 78 9

Groups 1-5 intact/ Sham operation group, no significant changes SFI the data were not included in this figure.

SFI values was about -90 severely impaired of SFI at 7 days (motor function) after crush injury

Discussion I

Grade 2: Axonotmesis (Seddon and Sunderland)

Crush + normal saline treated group

SFI Values in this study shows normal walking pattern in footprint test after the first month which is similar to several authors’ results (Oliveira et.al2001,Gudemez et.al 2002) While some author found the full recovery at the third and fourth week. (Bridge et.al 1994,Xavier et.al 2012)

Plasma MDA Result

*P< 0.05 represents significant difference vs other groups

5 6 7 8 99 8765

56 78 9

1

1 1

*Day 15 Day 30

56 78 9

1

5 6 7 8 99 87651 1

Normalwallerian degeneration occur 3-7 Day after crush injury

MDA level was high initially and then gradually declines

Bain et,al1989&Nam et,al.2010Day 7

Result

6 7 8 9

6789

*P< 0.05 represents significant difference vs. crush + normal saline group

NS

Normal HistopathologicalP< 0.05 represents significant difference vs crush + normal saline group

Histological analysis

Day 30

Presenter

Presentation Notes

For the H&E straning in group 1, no histopathological changes were observed

Histological analysis

Day 30

Edema (*)

Vacuolization (+)

Myelin ellipsoids(>)

B c

ED

Axon regeneration

Gaudet et.al 2011

Group SFI Day 1,7,14,21,28

Plasma MDADay15,30

Walleriandegeneration

1. Intact+NSS

2. Intact+Crocin 5 mg/kg

3. Intact+Crocin 20 mg/kg

NS No changes

4. Intact+Crocin 20 mg/kg5. Sham surgery+NSS

NS

6. Crush+NSS P< 0.05 increased

on day 15, but not on

day 30

Swelling of Myelin

sheet,

Vacuolization and

Myelin ellipsoids

7. Crush+Crocin 5 mg/kg NS NS

8. Crush+Crocin20 mg/kg P< 0.05 P< 0.05 Changes

9. Crush+Crocin 80 mg/kg On day 15*

*

*

Summary Result

Conclusion

MDA levels

Histological changes in sciatic nerve: Swelling of Myelin sheet,Vacuolization and Myelin ellipsoids

Sciatic nerve crush injury

Decreased free radicalsSFI Improved motor function

Wallerian degeneration

Crocin

A potent antioxidantNeuroprotection

Sharing Opinions

Nerve crush injury Grade 2: Axonotmesis

Positive control group as Ascorbic acid or Tocopherols , which makes this study more reliable

Blood collection: tail vien

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Sharing Opinions

Prevent risk for sciatica

Acknowledgements

Thank you!

Contact Address: Prof. Somchai Doe Tel:Email: www.kku.ac.th

Nerve Crush Injury

Axon degeneration

Sciatic Nerve Crush Injury

Microtubule disassembly

Axon swelling

Axon fragmentation

Removal of remnant

Wallerian degeneration at distal stump :Schwann cells (SC)& Macrophage

Ischemia

Cell damaged:damages cellular

proteins, DNA, and the plasma

membrane

Release of more free radicals

Reperfusion

Reintroduces O2

White blood cells

Release a host of inflammatory factors :

interleukins (IL-1 )

Free radicalformation

Reduction in oxygenAnd energy substrates

Caspase dependent pathway

Apoptosis

E.protease & phospholipase

Crocin

crocin enhanced functional recovery after sciatic nerve

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