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Damaged Neocortical Perineuronal
Nets Due to Experimental Focal
Cerebral Ischemia in Mice, Rats and
Sheep
AUTHORS
Wolfgang Hartig, Binaca Mages, Susanne Alithe, Bjorn Nitzsche, Stephan
Atlmann, Henryk Baethel, Martin Krueger and Dominik Michalski
Journal
Frontiers
In Integrative Neuroscience
PRESENTED BY:
Ashutosh B Mahale
M.Pharm (Pharmacology)
Introduction
Neurovascular Unit
•Ischemia displays largely decomposed and nearly erased PNs in
infracted neocortical areas that were demarcated by up-
regulated immunoreactivity for vascular collagen IV
Perineuronal net
Excitotoxicity
Hypoxia
ATP Supply
Reduced
K+ outside
Calcium entry
Glutamate
release
NMDA
Receptor
Binding
Excess Calcium Inside the cell
Change in
permeability
Cytochrome - c
Caspase-9
Caspase-3
Cell Death
Objective
• Ischemia related alterations of PNs as well
as associated neocortical glial and vascular
alterations in the models of:
-pMCAO in mice
-thromboembolic MCAO in rat
-electrosurgicaly induced pMCAO in sheep
Materials and Methods
1. 3 Test animals
• 5 C57BL/6 mice 25g of body weight
• Wistar rats of about 300g body weight
• 3 Male adult sheep
2. Models
• pMACO in mice
• Thromboembolic MACO
• Electrosurgically induced focal cerebral ischemia
3. Tissue preparation
INDUCTION OF FOCAL CEREBRAL
ISCHEMIA IN MICE
Anesthesia : Etomidate(33mg/kg i.p.) +Lidocaine
Silicon -coated 6-0 monofilament
Right sided pMCAO
Figure:- Surgical procedure of middle cerebral artery occlusion
RATS
Anesthesia:- Isoflurane 2-2.5%+70% N2O/30% O2
A PE tube was inserted into middle cerebral artery
Small amount of blood was withdrawal
Warm at 37⁰C for 2hr
store at 4⁰C
Prepared a blood clot of 45mm
Inserted with small volume of saline
SHEEP
• Anesthesia : ketamine (4mg/kg) +Xylazine (0.1mg/kg) +
Diazepam (0.2 mg/kg ) i.v
• Procedure :
Left temporal lobe was incision
trepanation 10000rpm with 6mm barrel
Dura matter was incised
Electrosurgical coagulation using neurosurgical bipolar
forceps
CONFIRMATION OF ISCHEMIA
• RATS AND MICE :
- Neurobehavioral deficit : Menzies score (min 2 points )
• SHEEP : - Visible ischemic lesion in magnetic resonance
tomography
- Done after 2 weeks
Tissue Preparation
• Rat and Mice :-
Forebrains were cut with freezing microtome
30µm thick section 10 series
• Sheep:-
- 10mm thick corneal slices prepared
photographed and immersed 4% buffer
formaldehyde for 14 days
- The slices cut at 40µm thickness using
freezing microtome
Perineuronal net ppt

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Perineuronal net ppt

  • 1. Damaged Neocortical Perineuronal Nets Due to Experimental Focal Cerebral Ischemia in Mice, Rats and Sheep AUTHORS Wolfgang Hartig, Binaca Mages, Susanne Alithe, Bjorn Nitzsche, Stephan Atlmann, Henryk Baethel, Martin Krueger and Dominik Michalski Journal Frontiers In Integrative Neuroscience PRESENTED BY: Ashutosh B Mahale M.Pharm (Pharmacology)
  • 2. Introduction Neurovascular Unit •Ischemia displays largely decomposed and nearly erased PNs in infracted neocortical areas that were demarcated by up- regulated immunoreactivity for vascular collagen IV Perineuronal net
  • 3. Excitotoxicity Hypoxia ATP Supply Reduced K+ outside Calcium entry Glutamate release NMDA Receptor Binding
  • 4. Excess Calcium Inside the cell Change in permeability Cytochrome - c Caspase-9 Caspase-3 Cell Death
  • 5. Objective • Ischemia related alterations of PNs as well as associated neocortical glial and vascular alterations in the models of: -pMCAO in mice -thromboembolic MCAO in rat -electrosurgicaly induced pMCAO in sheep
  • 6. Materials and Methods 1. 3 Test animals • 5 C57BL/6 mice 25g of body weight • Wistar rats of about 300g body weight • 3 Male adult sheep 2. Models • pMACO in mice • Thromboembolic MACO • Electrosurgically induced focal cerebral ischemia 3. Tissue preparation
  • 7. INDUCTION OF FOCAL CEREBRAL ISCHEMIA IN MICE Anesthesia : Etomidate(33mg/kg i.p.) +Lidocaine Silicon -coated 6-0 monofilament Right sided pMCAO
  • 8. Figure:- Surgical procedure of middle cerebral artery occlusion
  • 9. RATS Anesthesia:- Isoflurane 2-2.5%+70% N2O/30% O2 A PE tube was inserted into middle cerebral artery Small amount of blood was withdrawal Warm at 37⁰C for 2hr store at 4⁰C Prepared a blood clot of 45mm Inserted with small volume of saline
  • 10. SHEEP • Anesthesia : ketamine (4mg/kg) +Xylazine (0.1mg/kg) + Diazepam (0.2 mg/kg ) i.v • Procedure : Left temporal lobe was incision trepanation 10000rpm with 6mm barrel Dura matter was incised Electrosurgical coagulation using neurosurgical bipolar forceps
  • 11. CONFIRMATION OF ISCHEMIA • RATS AND MICE : - Neurobehavioral deficit : Menzies score (min 2 points ) • SHEEP : - Visible ischemic lesion in magnetic resonance tomography - Done after 2 weeks
  • 12. Tissue Preparation • Rat and Mice :- Forebrains were cut with freezing microtome 30µm thick section 10 series • Sheep:- - 10mm thick corneal slices prepared photographed and immersed 4% buffer formaldehyde for 14 days - The slices cut at 40µm thickness using freezing microtome