CNS Models in Rodents

CNS models in rodents: What can you expect from your experimental pathologist?

Dr. Emmanuel Loeb

Introduction

Three major parameters are important for accurate histopathological results in CNS models:

  • Tissue preparation for representative lesion exposure and analysis.
  • Choice of histological tools for measuring your desired end points.
  • Accuracy of the analytical process.

An example of a Stroke (MCAO) study in a rat model.

Tissue preparation for representative lesion exposure and analysis

Tissue harvest and fixation:

  • For fluorescence analysis, performance of brain perfusion prior to brain / spinal cord harvest.
  • Harvesting the organs by valid procedures to avoid artifacts.
  • Fixation of organs in 4% PFA and storage at 4°C.
  • Sample labeling and administration.

Brain, localization 1, striatum                                         Brain, localization 2, dorsal hippocampus

Brain, localization 3, cerebellum and                                      Brain, optional localization 4,

medulla oblongata                                                                       cerebrum and pons.

Abbreviations used in the above images:

Ac: anterior commissure, C: cerebellum, Cc: cerebral cortex, Cca: corpus callosum, H: hypothalamus,

Hc: hippocampus, Mo: medulla oblongata, O: optic chiasm, P: pons, Sr: striatum, T: thalamus

 

Tissue preparation and trimming

  • Preparation of the appropriate cross­section in the organ for representative exposure.
  • Assurance of having the relevant anatomical structure in the same position for all samples.
  • Use of brain matrix system for this purpose.
  • Replacement of all cross­sections for all animals in the same block position.

Tissue processing and slide production

  • Use of high­standard tissue processor with an accurate program for each tissue type.
  • Highly trained technician for block production and sectioning.
  • Production of top­quality histological slides for interpretation and analysis.

The choice of histological tools for measuring your desired end­points

Common stains and IHC markers used in CNS models

Choice of the best tool depends on the scientific aim and the relevant endpoint.

  • H&E: Used for general morphology and quantification of lesions.
  • Demyelination: MBP for myelin, Olig­2 for total oligodendrocytes, NG2 for young oligodendrocytes.
  • GFAP: for Astrocytes
  • Iba­1: For Microglia
  • NeuN:For neuron count
  • ChAT:For motor neurons
  • NF: For total neuron fibers
  • TH: For Dopamine­/norepinephrine­ producing cells (PD)
  • beta­Amyloid, Tau­P: For Protein products (AD)

Stroke (MCAO) study in a rat model. Left intact hemisphere, right affected side. Note the loss of myelin and increase of microglia.

Accuracy of the analytical process

Analytical process

  • Double­blind examination by an experienced pathologist
  • Semi­quantitative vs. morphometry analysis
  • Image analysis•Whole­slide scan (digital slides)
  • Advanced digital analysis•Accurate data
  • Avoid bias between pathologists

Ku­80 for human cells in treated brain infarct in a rat (MCAO) model.

TH (Tyrosine Hydroxylase) measurements in the SNc (substantia Nigra pars Compacta).

MBP quantification using segmentation analysis.

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