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 by Rujikan Chaisanam Neuroscience Program, Khon Kaen University
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Crocin Enhanced Functional Recovery after Sciatic Nerve Crush Injury in Rats
E.Tamaddonfard, A. Abbas Farshid, E. Ahmadian, A. Hamidhoseyn
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.
(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