Neuropathology

1. Neuropathology Strategies for conducting research with postmortem human brains

2. What neuropathologists do! • Perform autopsies to examine: • CNS [brain, spinal cord]; PNS [peripheral nerves, end-organ nerves]; Skeletal muscle; Eyes; Pituitary gland • Diagnose frozen section (immediate) and paraffin-embedded surgical biopsies ± EM, IF, IHC • Correlate neuropathology with clinical abnormalities • Bank postmortem and surgical biopsy specimens • Communicate results with family members • Drum up business

3. Steep Decline in Autopsy Rate Since 1970s Over the Past 35 Years, Far Fewer Autopsies Done on People Who Died of Disease

4. Translational Research

5. Macroscopic Examination of Brains Set-up • Large bucket with 3-4 volumes of fixative • Tissue banking kit (if needed) • Vials, jars, cassettes for non-brain and spinal cord samples • Proper protective wear- universal precautions. • Dissecting instruments; recording devices, camera In Advance • Confirm consent for removal and extent of postmortem exam • Review clinical records or discuss case with physician • Review underlying systemic diseases • Determine if case for routine diagnosis or research

6. Essential Stuff

7. Initial Examination • In situ examination of skull, dura mater, vessels, sinuses • Removal and macroscopic examination of the fresh brain, including weight • Ventricular fluid harvest through lateral ventricle • Removal and macroscopic examination of the spinal cord • Removal of eyes, skeletal muscle, peripheral nerves (sensory, motor and mixed)

8. External Examination Ventral Surface: Circle of Willis Ventral Surface: Cranial nerves Dorsal Surface

9. Extra stuff: The complete exam

10. Immediate assessments • Dura mater • Venous sinuses • Epidural and subdural surfaces • Leptomeninges • Clarity and color • Hemorrhages, neoplasms, exudates • Cranial nerves • Brain • Symmetry—gyralpattern; weight • Atrophy—distribution, severity • Swelling—acute injury or metabolic insults • Focal lesions—trauma, neoplasms, hemorrhage, exudate • Spinal cord, eyes, muscle, nerves

11. Dura Mater

12. Dura Mater

13. Subdural Hematoma

14. Cerebral Atrophy in AD

15. Routine Diagnostic Studies • Fix brain with spinal cord [2 weeks]-suspended in: • 10% formalin (RT) • 4% paraformaldehyde (4°C) • Histofix [or other proprietary formulations] (RT) • PLP (4°C) • Rinse over night in running cold tap water • Re-weigh fixed brain • Perform external examination • Cut brain and spinal cord using a standardized protocol

16. Knowledge of Real Estate

17. Standardized brain sampling

18. Parkinson’s Neuropathology Pallor of Substantia Nigra and Locus Ceruleus in midbrain and pons; loss of dopamine and norepinephrine producing neurons in brainstem

19. Microscopic Examination • 16-20 standardized blocks of tissue from: • Specific brain regions • Spinal cord • Eyes (retina) • Skeletal muscle • Peripheral nerves • Luxol fast blue, hematoxylin and eosin (LHE) stain • Silver impregnation stains, e.g. Bielschowsky • Congo red • Other histochemical stains

20. Temporal Lobe Asymmetrical Degeneration Right Left

21. Temporal Neurodegeneration

22. Amyloid Angiopathy

23. Substantia Nigra

24. SubstantiaNigra: Parkinson’s Disease

25. Extended histopathological analyses • Immunohistochemical staining to identify specific abnormal proteins accumulated due to misfolding and ubiquitination • Phospho-tau • Ubiquitin • TDP-43 • /β crystalline • -synuclein • β-amyloid (Aβ-AβPP) • Replaces histochemistry, EM, fluorescence

26. Amygdala-AD Neurodegeneration

27. Cerebral Amyloid (Abeta) Accumulation in AD

28. Pick’s Neurodegeneration

29. Alzheimer+ Causes of Dementia

30. Research Donation Cases • Standardized protocol for fixing and freezing brain tissue, spinal cord, etc: • Surface map sampling • Slice brain, sample specific structures • Hemi-section brain—freeze one half ** • Hemi-section brain, slice one half, freeze slices** • Snap freeze small tissue samples in isopentane or methanol cooled with dry ice or use liquid nitrogen • Freeze brain slices on metal plate cooled with dry ice. • Store frozen tissues at -80°C or lower in alarmed freezer

31. Harvard Tissue Resource Center

32. Modern Tissue Freezing Center

33. Research Donation Cases • Additional approaches for pathological studies • EM fixative • MRI-magnetic resonance imaging • MRS-magnetic resonance spectroscopy • Newer approaches • RNA-Later for gene expression and profiling • Cryopreserve for later culture • Transfer to culture medium for immediate culture • Extract subcellular components from fresh tissue • Cell isolation; FACS, FLOW • Fluorescence in situ hybridization (ISH)

34. Tau Protein Insulin Deficiency Insulin Resistance •  GSK-3β •  CDK5 •  P38 MAPK • JNK •  PP2A P P P P P Hyper-phosphorylated Tau ROS U U U U U Mis-folded, hyper-phosphorylated, ubiquitinated Tau U U U U Insoluble fibrillar Tau aggregates Neurofibrillary tangles Dystrophic neurites Neuropil threads Synaptic disconnection Oxidative stress Neuro-inflammation Mitochondrial dysfunction Cell death

35. Diabetes Mellitus Steatohepatitis Obesity Peripheral Insulin Resistance Type 3 Diabetes Brain Insulin Deficiency CNS Metabolic Dysfunction CNS Oxidative Injury Brain Insulin Resistance Tau Pathology • AβPP • Expression • AβPP -Aβ GSK-3 β Apolipoprotein E ϵ4 Presenilin Mutation Stress PI3K-Akt Oligomers ADDLs Plaques

36. Technology Has Arrived: We Haven’t

37. Limitations of postmortem human brains for research • Postmortem decomposition (autolysis) • Time and temperature dependent (delays in obtaining consent and transporting the body • Underlying systemic diseases can confound or complicate the neuropathology • Inaccurate clinical assessments; long interval between last exam and death • Lack of any available history • Prion disease scares • Improper or suboptimum tissue handling and storage • Non-uniform banking and sampling practices over time