005 Neurocytological and Imaging Techniques Flashcards
Cationic Stains
(AKA Nissl Stains)
Stains that act as a weak base
Stain acidic cellular components
• Including nucleic acids
Good at identifying neuron cell bodies
• Show nuclei, nucleolus and Nissl Substance
Do NOT stain processes well (i.e. axons and dendrites)
White matter stains dark; gray matter stains light
Myelin Stains
Bind to the phospholipids of myelin
Hydrophobic stains (lipid based)
Good at staining myelinated processes (i.e. nerve fibers)
• Commonly used to show tracts in the CNS
Do NOT typically stain neuron cell bodies
*usually look like a photographic negative of a Nissl stained section
White matter stains light; gray matter stains dark
Golgi Stains
Selectively stain only a few cells in a section (about 10%)
Mechanism is still not entirely understood
The cells that do stain are completely filled
• Give the full architecture of only a few cells in a section
o Histochemical/immunocytological stains
Histochemical
• Uses a chemical
Immunocytological
• Uses an antibody
Selectively shows a specific structure/substance within a cell
A dye is attached to the chemical/antibody so it can be viewed
Reveals patterns NOT seen with other stains
Reduced silver stains
Metallic Stain
Have affinities for certain components in cells
Most commonly used to stain glial cells in the nervous system
Appear black in the light microscope because the metal salts deposited in tissues block light
• describe the common experimental methods used for determining connections in the nervous system,
degenerative methods, axonal transport methods, and lipophilic tracers
Degenerative methods for determing connections
Researchers make a lesion in a known location then look to find where signs of degeneration are located
• This allows inference as to where the rest of the nerve cell is
• The cell body that shows degeneration after an axon is cut can be inferred to be where the axons come from
This works because when a nerve cell, especially an axon, is injured the entire nerve cell shows signs of degeneration
Retrograde Degeneration
• The cell body located proximal to an injured axon undergoes changes, called axon reaction
• The degeneration is going backward compared to the direction of nerve impulses
• The changes can be seen with Nissl stains
Anterograde degeneration
• Wallerian degeneration
o When an axon is injured, the axon distal to the injury undergoes degenerative changes
• The degeneration is going in the same direction of the direction of flow of nerve impulses
• The axons that show degeneration after an axon is cut can be inferred to be where the axon is going
o The most distal place where degeneration occurs is where the axon synapses
Axonal transport methods for determining connections
Tracer is injected into a known location
Cells take up the tracer and move the tracer within the cell via axonal transport
Causes NO damage to the cell
Wherever the tracer is found can be inferred to be where the cell is
anterograde axonal transport methods
- Uses tracer that is absorbed by the cell body and transported to the axon terminal
- Used to infer where axons go (synapses)
retrograde axonal transport methods
- Uses tracer that is absorbed by axon terminal and transported back to the cell body
- Used to infer where axons come from (cell body)
Techniques to make tracers viewable under a microscope
• Autoradiography
o ANTEROGRADE method ONLY
o Radioactive tracer
o Seen by the cell as a nutrient
o Injected into a known region
o Cell bodies absorb the tracer and transport it to their axon terminals
o Sectioins of tissue are then coated with a sensitive emulsion
Developed like a photograph to make a viewable image
• Fluorescent dyes
o RETROGRADE method ONLY
o Substances that fluoresces under a known wavelength of light
o Dye injected into a known region
o Axon terminal absorb the dye and transport it to the cell body
o Sections are examined in the specific wavelength of light
• Horseradish peroxidase (HRP)
o BOTH anterograde and retrograde methods
o Natural enzyme found in horseradish plants
o Absorbed by nerve cells, both at the cell body and axon terminals
o Injected into known locations
o HRP is axonally transported
o HRP is located by an immunologic assay
o HRP can be transported across a synapsed to the next neuron
Allows for tracing of neurons in circuits
Lipophilic tracer method for establishing connections
E.g. DiI
BOTH anterograde and retrograde methods
Dyes that diffuse through lipid moieties (e.g. myelin) of cells
Hydrophobic so it does NOT leak into surrounding tissues
Dye is introduced and give time to diffuse
Usually visualized with fluorescent methods
The tissue does NOT have to be alive
• Will work when alive because they are nontoxic
• Tissue is commonly harvested from the animal first
• Lipophilic tracers will work even after the tissue has been fixed
o Fixation=process to prevent putrefaction of the harvested tissue by denaturing/crosslinking proteins
o Fixation only changes proteins, not lipids
o Skull plain film x-rays
Standard x-rays
Give information about the skull (i.e. skull fractures)
Can sometimes give indirect information about the CNS
SOME ionizing radiation is delivered to the patient
o Cerebral angiography
Radiopaque dye is injected into blood stream
Standard exrays are taken at appropriate times to view blood vasculature
Gives information on the status of the blood vascular systems (aneurysms)
SOME ionizing radiation is delivered to the patient
o Pneumoencephalography
Air injected into spaces that are normally occupied by the CSF
An x-ray is then taken
Shows the ventricles of the brain and can check for blockage of CSF flow
NOT used that often anymore
Can causes very bad headaches
SOME ionizing radiation is delivered to the patient
o Computerized (axial) tomography (CT (CAT))
Multiple x-ray images are put together by computers to create images of sections down to 1 mm thick
Radioopaque media injected to enhance contrast (e.g. to view blood vessels)
Excellent views of hard structures, like bone, obtained
Quite a bit of detail of soft structures obtained
Relatively inexpensive compared to other processes showing the structure of soft tissues
Quite a bit of ionizing radiation is delivered to the patient
o Magnetic Resonance Imaging
Uses strong magnetic field to cause atoms (usually Hydrogen atoms in water) to alter their spin
Change in spin creates a signal that is detectable
A computer then creates images of sections down to about 3 mm thick or less
Unlike x-rays, which are based on true densities of tissues, MRIs are based on relative amounts of water
NO ionizing radiation delivered to patient
Excellent detail of soft tissue
EXPENSIVE
Functional MRI
Based upon the fact that oxygenated and deoxygenated hemoglobin have different magnetic properties that can be detected by an MRI
The images formed are based upon relative amounts of oxygenated and deoxygenated blood
Tells the relative metabolic activity of various tissues
• More active means more blood means more oxygenated blood
o Positron emission tomography (PET)
Radioactively labeled tracers injected into patient
• Usually 2-deoxyglucose
Tracer taken up by cells but not metabolized
Positrons emitted by radioactive tracer are detected and used to create an image
Requires an on-site cyclotron
• VERY EXPENSIVE TECHNIQUE
Image based on relative amount of tracer taken up by cells
• Can infer metabolic activity of cells
• More active means more glucose taken up by cells, but the cells cannot use the tracer
SOME ionizing radiation delivered to patient
o Single photon/positron emission computerized tomography (SPECT)
Uses a radioactively labeled tracer (similar to PET scans)
Can infer metabolic activity of the tissues (similar to PET scans)
Technique NOT too expensive
Images do NOT have great resolution
SOME ionizing radiation delivered to the patient
• describe some limitations of the current state-of-the-art of imaging techniques
o They are slow
Measured in the realm of minutes
Neural events are in the realm of milliseconds
Can measure/infer metabolism but NOT cognitive function
• describe the axon reaction
o name for changes in the neuron that occur proximal to the injury (on the soma side)
o In larger neurons, if only the axon is damaged, the cell can sometimes survive through a series of steps
o Several steps
Axon degenerates to the next proximal Node of Ranvier
Chromatolysis occurs about 24-48 hours after injury
• Nissl substance changes into finely dispersed granulations
• Rough ER breaks up with an increase in the number of free ribosomes
• Represents the protein producing machinery gearing up to heal the cell
The nucleus becomes eccentric in the soma
The soma swells to reach a maximum about 10-20 days after injury
The nucleolus enlarges which implies increased rRNA production
• describe wallerian degeneration
o the name for changes in the nerve fiber distal to the injury
o several steps
the axon swells and becomes irregular in the first 24 hours
the axon then breaks into fragments after about 3-5 days
during this same time (1-3 days after injury) the myelin changes into short elliptical segments
Next Schwann cell proliferation begins to occur
• The new Schwann cells fill the space vacated by degenerating axon of the endoneurium
While all of this occurs, the debris of the axon and myelin sheath is phagocytosed by histiocytes (hisitocytes are connective tissue macrophages)
Bands of von Bugner form
• Tubular formations of endoneurium filled with Schwann cells in the distal stump of a degenerated nerve
Axon regeneration in the PNS
Axon regeneration can occur if
• Either the connective tissue is NOT disrupted with injury
• The ends of the severed nerve are re-apposed (e.g. surgically)
Stages
• Axons start to regenerate from the last intact internode of Ranvier and invade the region of the bands of von Bunger by about day 4
• The tip end of the regenerating axons form growth cones that guide the axon as it continues to elongate
• Axons continue to grow and hopefulle follow endoneurial tubes at a rate of 1.5 mm per day
o If growing axons miss the bands of con Bunger they may continue to grow and rollup to form whorls that become neuroma (which are pain producing)
• The axons become remyelinated (if myelinated in the first place)
*unmyelinated neurons have a very low rate of successful regeneration
At best, regenerated nerve fibers function no better than 80% as efficient as originals
Axon regeneration in the CNS
Starts similarly to the PNS, but there is effectively NO regeneration of CNS axons
Stages
• Astrocytes invade the region to form an astrocytic scar
• Wallerian degeneration occurs in the regions of fibers distal to injust but with oligodendrocytes instead of Schwann cells
• Microglia phagocytose the debris
o Instead of histiocytes because CNS does not have connective tissue
• Axons begin to regenerate by forming growth cones, but stop growing after about 2 weeks
o Appears to be a signal from oligodendrocytes that stops the growth
• describe neural plasticity
o The ability of the nervous system to change to allow at least some functional recovery to occur, even without anatomical regeneration
o Involves the taking over of the functions of the damaged region by other regions that have NOT been damaged
Done through a reorganization of connections on the synaptic level
Accompanied by some synaptic structural changes
o Example
Rehabilitation of patients after a stroke
