Download felix may 2nd year neuroscience Investigation into the response to

felix may
2nd year neuroscience
Investigation into the response to injury in rat brain to a chemical insult, using
Immunohistochemistry
Abstract
Glial cells constitute a large part of the central and peripheral nervous systems, occurring in greater
numbers to neurons, and are usually present wherever neurons are. We aimed to use
immunohistochemistry to track the changes in astrocytes and microglia after a chemical injury over a
period of 28 days. An experimental model for brain injury was used: 3-chloropropanediol is a neurotoxin
known to produce specific brain lesions in the colliculi and red nucleus, as well as a transient weakening
of the blood-brain barrier. After infusing the neurotoxin the rats were sacrificed at intervals to allow
periodic observations of the progress of the brain’s response.
Astrocytes express glial fibriallary protein (GFAP) whilst microglia express CD11b, which will be the
markers for these two cell types. A two step indirect method of immunological staining was used to
visualise these cells. Primary monoclonal antibodies raised in mice against GFAP and CD11b and
secondary antibody raised in goats (goat anti mouse - GaM) conjugated to horseradish peroxidase (HRP)
were used. Visualisation was then done with the chromogen 3,3-diaminobenzide (DAB).
Our observations showed that 3-chloropropanediol produced a localised lesion in the inferior colliculi,
seen as a zone with few resident cells and much debris. As time progressed the lesion was re-infiltrated
first by microglia and then by astrocytes. There was an increase in the abundance of both cell types and
the intensity to which they stained.
After a chemical insult the rat brain responded by activation of glial cells which proliferated into the
damaged region, forming a scar.
Introduction
Glial cells constitute a major part of the central and peripheral nervous systems, there being about 30
times more glial cells than neurons. Glia are non-neuronal cells that support neuronal function by
optimising the local environment and providing trophic factors and nutrients, having a homeostatic
function. Unlike neurons glia continue to proliferate throughout life, especially during the reaction to
injury. Without glia the nervous system could not function.
Astrocytes (and other glia) are intimately involved in the formation of new processes from neurons. The
infiltration of astrocytes into an area preceeds the growth of neural processes. By observing the activity
of astrocytes an insight can be gained into the initial stages of repair and recovery after brain injury.
Microglia are the brain’s resident immune cells, and are mobilised in times of crisis to re-establish
conditions for glial and neural growth.
There are four main types of glia:
Astrocytes are the most abundant type of glia. They have many cellular processes and so appear as
star-like forms. Astrocyte processes grow around neurons and capillaries, forming ‘end feet’. The
secretion of trophic factors from astrocytes, and also pericytes, induces the formation of tight-gap
junctions between the capillary endothelial cells. This is the basis for the blood-brain barrier. The BBB is
both a physical barrier restricting the passage of large molecules, and also an enzymic barrier that acts on
many chemicals before they can pass through. Astrocytes also act to regulate local ion concentrations,
and to take up substances, most notably neurotransmitters, from the intracellular space. Lactate, the
primary energy substrate of neurons, is produced by astrocytes from glucose.
Microglia, although not originating from ectodermal tissue, are also classed as glia. These small cells aid
neurons by acting as specialised macrophages, digesting and removing debris and invaders. They enter
the central nervous system from the circulation during embryogenesis.
Like Schwann cells in the periphery, oligodendrocytes myelinate neurons in the CNS, creating white
matter. Once myelinated the speed of axon action potential conduction is greatly increased, its maximal
distance extended, and its influence on collateral neurons reduced. Schwann cells myelinate a single
axon, while oligodendrocytes myelinate multiple axons.
Ependymal cells line the ventricles and are found in the subarachnoid space. They possess cilia that
encourage the flow of CSF, and form part of the choroid plexuses that produce CSF.
Methods
Rats were infused with 3-chloropropanediol and sacrificed at incremental time points. The midbrain was
the removed from the animal, sliced on a microtome, and prepared for mounting on slides.
Fresh frozen tissue sections 30m thick were taken out of cold storage at -80C and were air dried for 10
minutes. After drying the tissue was encircled with water repellent.
Five slides were prepared: two for GFAP binding, two for CD11b binding, and a negative control using
mouse antibody to control for non-specific binding of mouse serum and the secondary antibody.
The slides were fixed in pure ethanol for 10 minutes, then flooded with PBS, and then washed four times
with buffer-1 (1% BSA and 0.2% Tween-20 in PBS).
After removing the buffer normal goat serum was applied to all the slides, then left to incubate for
15mins.
The NGS was then removed and 250l of diluted primary antibody was added to the slides and allowed
to incubate for 30 minutes. The concentrations of antibodies were as follows: GFAP 13g/ml, CD11b
12g/ml, mouse Ig 10.6g/ml.
After the 30 minutes the primary antibodies were washed off thoroughly using buffer-1. Then 250l of
the secondary antibody - goat anti mouse (GaM), conjugated to horseradish peroxidase - was added; its
concentration was 10g/ml.
The slides were then stained with the chromogen DAB to visualise the secondary antibody binding areas.
After being incubated for 10 minutes in 2.0mg/ml DAB diluted in PBS with 0.003% H 2O2, the slides
were washed with distilled water.
A small amount of mountant media (Citiflour AF1) was then applied to a coverslip which was then
carefully placed onto the slide.
The slides were then ready to be viewed.
*Buffer-1 contains bovine serum albumin to reduce non-specific antibody binding on the slide.
Results
Observations were taken of each of the five slides under low (x5) and high (x40) power objectives.
GFAP at 8 days (fig.2) compared to GFAP at 0 hours(fig.1):
The 0 hr slide showed regular staining throughout the tissue. Astrocytes could be seen clearly under high
magnification, having distinct cell bodies and processes, with a visible nucleus.
In the 8 day slide a large lesion was present in the region of the inferior colliculus, in which some faint
debris were visible. Around the border of the lesion there was dark staining visible at low magnification,
darker than the staining elsewhere in the slide, and darker than that in the 0 hr slide. Closer inspection
under high magnification revealed a dense infestation of astrocytes. These astrocytes were denser and had
more processes than those elsewhere on the 8 day slide, and more so than on the 0 hr slide. The processes
and cell bodies were also more tangled and interweaved on the border of the lesion compared to
elsewhere on either slide. Their processes were also comparatively ill-defined and swollen compared to
the slim and discreet processes seen in the 0 hr slide.
The nuclei of these infesting astrocytes were hard to distinguish from the cell body, but looked large
compared to those seen in the 0 hr slide.
CD11b at 8 days (fig.4) compared to CD11b at 0 hours (fig.3):
In the 0hr slide a faint even scattering of staining was visible throughout the tissue under low
magnification. Comparing this to the 8 day slide, in the latter there was a massive dense patch of staining
around the area of the inferior colliculus, where the lesion was observed in the 8 day GFAP slide.
Closer observation under high magnification showed that the 8 day slide had a dense population of
microglia in the site of the lesion, most dense in the core and thinning out towards the edges. The quality
of the cells had changed between the two slides - the 8 day slide had microglia closer together and with
more densely stained bodies, with a matrix of fuzzy staining between them. In the 0 hr slide the glia were
relatively dispersed and their cell bodies were more distinct.
Negative control - mouse Ig (fig.5):
There was minimal staining, an almost transparent slide, with the borders of the tissue just visible. Two
slits were visible, although these looked like mechanical damage to the tissue, presumably artefacts of the
mounting process.
The entire group pooled basic observations on the abundances of each cell type in their slides. The results
are displayed in the original table:
0h
3h
6h
12h
h
18h
h
24h
h
48h
h
3d
4d
6d
8d
14d
dd
28 d
GFAP
+
+
+
+
+/-
+/-
+/-
-
-
-
+/-
+/-
++
CD11b
+
+/-
+/-
+/-
+
+
++
++
+
++
+
++
+
++
+
++
+
++
figure 6 - cell abundances over a 28 day time course
Discussion
Infusion of 3-chloropropanediol causes specific brain stem lesions in the rat. We observed the lesion in
the inferior colliculus over a time course of 28 days.
The presence of cells staining for GFAP (astrocytes) and CD11b (microglia) showed dramatic increases
in their number, and in the intensity of cell staining. This indicates that the injury caused an activation
and mobilisation of microglia and astrocytes.
The proliferation of glia is not homogenous in location or time. In our slides it was apparent that glial
activity was focused in and around the lesion site in the inferior colliculus. Some lesser activity was also
observed outside of the lesion site.
The group reports of cell abundances suggest that there is a biphasic increase in astrocyte abundance,
interjected by a transient decrease. Initially the astrocyte population is destroyed in the site of the lesion.
The astrocytes then proliferate at the edge of the lesion, re-infiltrating gradually.
The microglia appear to proliferate steadily but slowly, taking 48 hours to reach their peak abundance.
During this proliferative period the microglia infiltrate the lesion site and populate it, with the greatest
density in the centre of the lesion.
By 28 days after the infusion of the neurotoxin, there was still an apparent increase in the abundances of
microglia and astrocytes relative to the state at 0 hours.
Injury in an organism leads to inflammatory responses and the mobilisation of macrophages. In the CNS
the response is different to the rest of the body, with resident microglia taking the role of macrophages
and monocytes. When the CNS is damaged the microglia move out of quiescence to two possible active
states. One of these states involves proliferation and increased expression of surface proteins and
cytokines. If cell debris is present from necrotic neurons, microglia can also take on macrophage like
properties, forming lysosomes, secreting chemicals involved in phagocytosis and gaining a ruffled cell
membrane.
The increased presence of surface proteins (presumably including CD11b) and a ruffled membrane could
explain the appearance of the microglia in the 8 day slide.
The relatively small number of microglia in the CNS, compared to macrophages outside of the CNS,
means that the clearance of debris is significantly slower inside than outside of the CNS.
Secretion of tumour necrosis factor- and interleukin 1- and expression of the transcription factor Egr-1
in microglia occurs as part of the inflammatory response. These exert effects on vascular permeability,
degrading the blood-brain barrier temporarily.
While the blood brain barrier is compromised macrophages can infiltrate and contribute to the
inflammatory response.
The observations made from the group report of cell abundances are tenuous due to the subjectivity of the
grading. Using the same people to observe the relative presence of cells would help to improve the
reliability of the data. Furthermore a quantitive assessment, say by a machine, would prove even more
useful.
Observing all the slides directly would have been informative, although this was not possible due to time
constraints.
Further investigation could include analysis of post-transcriptional mRNA for specific markers such as
Erg-1 and C1q. This would permit a more detailed analysis of the localised and generalised cellular
reactions to brain injury.
A comparison of different types of brain injury and insult could also be interesting. For instance using
neurotoxins other than 3-chloropropanediol, or injecting inflammatory factors such as IL-1 and TNF
directly, or implanting active macrophages and microglia directly into brain regions. Using models of
ischemic and haemorrhagic injury would also be of use for comparison. Gene knockouts and the use of
anti-inflammatory agents could further reveal the contribution of individual factors to the whole response
to injury.
References:
Brain damage, brain repair - J. W. Fawcett, A. E. Rosser, S. B. Dunnett - Oxford University Press (2001)
Microglial activation and increased synthesis of complement component C1q precedes blood–brain barrier
dysfunction in rats - Nicholas J. Lynch, Colin L. Willis, Christopher C. Nolan, Silke Roscher, Maxine J. Fowler,
Eberhard Weihe, David E. Ray, Wilhelm J. Schwaeble - Molecular Immunology 40 (2004) 709–716