Immunohistochemical study of glial cell response to chemical brain insult in rats

URN:XXXXX 02/03/2010 University of Nottingham

Introduction

Immunohistochemistry is a scientific method used to track the distribution and localization of chemical markers, drugs, proteins, various pathogens and gene sequences in the tissue section; utilising a combination of immunoglobulin markers (antibodies). This imaging technique, widely practised in pharmacological and biomedical research, allows the visualisation of the pathways of cellular actions. It therefore aids early diagnosis of inflammatory diseases and neurodegenerative disorders. Immunohistochemistry uses two types of antibodies: primary and secondary. Primary antibodies are raised against a particular antigen in the tissue and are often unlabelled, while secondary antibodies are raised against primary antibodies and often contain a coloured marker. Visualisation of an antibody-antigen interaction can be done in a number of ways; one of them, conjugation of an antibody to an enzyme (secondary antibody) produces colour reaction detectable under light microscopy. Alternatively, the antibody can be tagged to a fluorescent marker such as fluorescein, rhodamine, or Alexa Fluor and examined using fluorescent microscopy. Although using two antibodies is not essential, increasing the level of selectivity ensures that the bioluminescent marker binds to the correct target molecule, allowing for accurate in-situ imaging of target cells and promoting reliability of results. In order to visualise glial reaction to chemical insult 2-step indirect analysis was performed. Polyclonal mouse antibodies (PMIgs) complimentary to GFAP or ED1 were used as primary antibodies. GFAP stain is used to determine the presence of glial fibrillary acidic protein, expressed by astrocytes and ependymal cells in the CNS. Despite its wide use as a biomarker, its function still remains unclear. The amount of GFAP expression is regulated by numerous factors, such as cytokines and hormones, with over-expression being common in astrocyte activation during brain injury. ED1 is a cellular marker specific for activated microglia, monocytes and macrophages – the resident immune system in the CNS. Goat anti-mouse antibodies conjugated with horseradish peroxidase were used as the secondary antibody (GaMIg-HRPs). Horseradish peroxidase is an enzyme, which produces colour after binding to GaMIgs. Abbreviations: 3-CPD, 3-chloro-1,2-propanediol; CNS, central nervous system; DAB, 3,3'-diaminobenzidine; ED-1, specific marker for activated macrophages/ microglia; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; HRP, horseradish peroxidase; mRNA, messenger ribonucleic acid; NGF, neuronal growth factor; NGS, normal goats serum; PBS, phosphate buffered saline Aims/Objectives

investigation of temporal course of microglia and astrocytes activation in response to chemical brain insult

understanding and practice of immunohistochemistry methods using two stains: GFAP and ED1

observation of morphological changes and distribution of activated glial cells under light microscopy

Methods

Prior to the practical

Rats were infused with 140mg/kg dose of 3-CPD and sacrificed at progressive time points. After the removal of the midbrain from the rat, the brain was sliced using a microtome and prepared for mounting on slides. Fresh 30µm thick brain slices were frozen at -80oC temperature, taken out and dried for 10 min, after which they were encircled with water repellent. Slides were dried for 30min prior to the experiment.

During the practical

Two slides were prepared in the class: (i) negative control, incubated with buffer and secondary antibody for detection of non-specific binding and (ii) positive control slide, incubated with both primary and secondary antibodies for detection of GFAP. Slides were fixed in ethanol for 10 minutes, then flooded with PBS (2x) and washed three times (5min each) with buffer-1 using transfer pipette. NGS was applied to both slides and left to incubate for 10 min. Slides were then incubated for one hour in the following manner: positive control slide was incubated in 250µl primary antibody (monoclonal anti-GFAP) and the negative control slide was incubated in 250µl buffer. After the break both slides were washed with buffer 1 and incubated in the secondary antibody (goat anti-mouse linked to HRP) for another 30min. After another set of washing the slides with buffer 1 (3x1min) and PBS (2xmin), they were taken to the DAB station, placed in a rack where staining trough in DAB was performed by a laboratory technician (5min). Slides were washed in dH2O, dehydrated and mounted in AMU for observation.

Results

(i) Examination of our own immunostained sections

GFAP staining results displayed in the Table 1 show the extent of non-specific binding in healthy brain sections observed under light microscope (x40 magnification). Negative control slide (a), stained without the primary antibody, showed ependymal cell staining around the walls of arteries in the arachinoid space. Positive control slide (b-e), stained with both primary and secondary antibodies, resulted in regular staining throughout the tissue. Non-specific staining of ependyma was recorded at positive control (Figure 1 b and c). No astrocytes were recorded in white matter (c). In the grey matter (d), astrocytes appearance is characterised as highly branched cells with slim processes and distinct cell body.

(ii) Examination of pre-stained rat brain sections

Figure 2 shows the appearance and abundance of astrocytes (GFAP) and microglia (ED1) in the inferior colliculus at different times of survival from injury. GFAP stained section, obtained 4 days after the insult with 3-CPD, shows a large lesion in the region of inferior colliculus in both hemispheres and dark staining around the border of the lesions compared to little or no staining in the core and surrounding tissues. Closer inspection under 40x magnification revealed the presence of dense stellate shaped astrocytes, more numerous in lateral regions of inferior colluculi (++) than medial (+/-). They appear larger, show more processes and are swollen compared to slim and delicate cells recorded on individually stained positive control slide.

ED1 staining, performed on the 6th day of cell reaction, showed dark staining in the area of inferior colliculus. Observation under higher magnification, revealed dense population of microglia abundant in the site of the lesion, denser in the core (+++) and thinning towards the debris (++). In the lateral region of inferior colliculus, most microglia observed are rounded in cell shape (however some irregular cells have been found), vary in size and are visibly scattered, while spaces between them are filled with fuzzy stained matrix.

Both slides show symmetry in cell distribution and morphology between two hemispheres.

Table 2 represents the class scores for changes in astrocyte and microglial cell distribution over 28 days of survival. GFAP staining does not show a clear relationship between the cell count and time of survival. There are little or no cells recorded in rat inferior colliculus from 0h to 28 days from insult comparing to the surrounding area, with exception of 3d and 4d where number of cells rises (+/++), after which cells disappear until day 28. Most of groups recorded stellate shaped astrocytes; although some rounded shaped cells (microglia) were found (6-8 days). ED1 staining, as opposed to GFAP, shows clearly the rising trend in the cell number with the time of survival. There is a significant rise in the cell numbers in day 2 (++), which continued until day 28 (+++) in both medial and lateral inferior colliculi. Most of the cells detected were of a rounded shape, with some stellate and mixed sections between 0hr and 18hr. Table 2 does not account for any differences of cell distribution between the hemispheres.

Discussion

This experiment demonstrates activation of astrocytes and microglia as the response to brain injury in the CNS. GFAP and ED1 markers revealed a dramatic increase in cell numbers in the lesion and certain morphological changes in both cell types.

3-CPD, applied during the experiment, is a neurotoxic agent commonly used as a model for neurotoxicity, due to its selective toxicity towards astrocytes in the inferior colliculi and red nucleus regions (Cavanagh & Nolan, 1993; Willis et al., 2004) allowing thorough examination of the nature of neurotoxic reaction cascade. It disrupts brain energy metabolism via catalytic inhibition of GAPDH (a polyfunctional protein that plays a crucial role in glycolysis) (Sirover, 1997), and inhibition of glutathione-S-transferase. No similar metabolic insult was observed in studies on 3-CPD resistant neocortex. Its effect can be antagonized by addition of pyruvate, the product of GAPDH (Sheline and Choi, 1998).

Astrocytes reaction to brain damage

Astrocytes are the most numerous non-neuronal cell type in the CNS (Tower and Young, 1973). They are divided into 3 major types according to morphology and spatial organization: radial astrocytes surrounding ventricles, protoplasmic astrocytes in gray matter and fibrous astrocytes located in white matter (Privat et al., 1995). They play variety of functions essential for neuronal survival such as: glutamate uptake, glutamate release, K+ and H+ buffering, water transport. They are involved in cell signaling through cleavage of neurotransmitters from the post-synaptic cleft and regulation of extracellular ionic concentrations. Astrocytes play two important roles in blood-brain barrier: 1) filter out nutrients essential for neuronal metabolism from blood capillaries via the endfeet and 2) release trophic factors (together with pericytes) into the blood to promote tight gap junction formation which is important for blood-brain barrier integrity.

Astrocyte-neuron reactions influence neurite outgrowth via the release of NGF, which is important for brain recovery during the post-injury period in cerebral ischaemia, hypoxia, trauma, brain hemorrhage, hypoglycemia and other insults. During injury within the CNS, astrocytes go through intensive morphological changes including heavy proliferation, heterogeneity and hypertrophy. Some astrocytes become phagocytic to ingest irreparable nerve cells.

Increased proliferation rate modifies the extracellular matrix promoting neurogeneration around the damaged region. Astrocyte secretions such as laminin, fibronectin, tenascin C, and proteoglycans are important modulators of neuronal development and are the component of glial scar. The elevation in cell number was not clearly present in class results due to limitations of the experiment explained in the separate paragraph. However, there was an increase number of astrocytes in the pre-stained section, four days after the insult confirms proliferation and migration of astrocytes at the edge of the lesion, re-infiltrating gradually to the core.

Heterogeneity is another important feature of astrocyte response to CNS injury. It depends on the nature of the injury, the proximity to the lesion and the microenvironment, leading to changes such as gene expression, increased secretion in the site of injury and modulating activity of other astrocytes. Astrocyte hypertrophy is a result of increased expression of glial fibrillary acidic protein, which leads to higher synthesis of cytoskeletal supportive structures to extend pseudopodia, and as a result increase in size. It is visible on the pre-stained slide, where glial cells appear larger and denser compared to positive control. Microglial reaction to brain damage

Microglia is a non-neuronal cell line, originating from monocytes (Ritter et al., 2006), which constitute 20% of the glial cell population in the CNS. Being the resident macrophages of the brain and spinal cord, they act as the first line of active immune defense in the CNS. (Gehrmann, Matsumoto & Kreutzberg, 1995). Due to restricted permeability of the blood-brain barrier, antibodies cannot enter the brain, thus microglia plays an important role in detecting foreign bodies, phagocytosis, and in acting as antigen-presenting cells activating T-cells. Their sensitivity to potassium levels allows them to recognize even small pathological changes in the CNS. (Dissing-Olesen et al., 2007). Their contribution to immune response also includes promoting inflammation and homeostatic mechanisms within the body by secreting cytokines and other signaling molecules.

Microglia are extremely plastic and undergo a variety of structural changes based on their location and current activation state. During inflammation, microglia become active, they take on an amoeboid shape and they increase their gene expression. This leads to increased production of numerous potentially neurotoxic mediators (Streita, 2006), high levels of which contributes to neuronal apoptosis in chronic inflammation (Friedlander, 1996). Previously mentioned plasticity of microglia explains their occurrence in three different states of activity: ramified (inactive) and two active states including phagocytic and Activated microglia begin proliferating and expressing various proteins on their cell membranes such as MHC-II and interleukins. The final activated stage, microglia are able to phagocytise antigens, but unlike resting microglia can then present the antigen to T cells within the CNS. These T cells can then produce antibodies for the antigen.

Experimental results show microglia in activated state, which is both confirmed by the dramatic rise in cell numbers in the lesion and the appearance of the cells on the slide prepared 4 days after the insult with 3-CPD. Extensive proliferation and stained matrix surrounding cells, which can account for increased secretion of various cytokines and inflammatory factors, show microglial anti-inflammatory response.

Limitations/ errors

As with most physiological experiments, certain discrepancies concerning the experimental method and procedural errors should be addressed. First of all, there is some extent of non-specific binding present in the negative control slide. This is due to either endogenous peroxidase activity in the brain or exogenous peroxidase binding to tissue membranes. This inconsistency requires subtraction of the staining in the negative control from the positive control in order to estimate the actual staining.

Secondly, frozen brain sections are fragile therefore pipetting needs to be performed at a low pressure to avoid scraping cells on the bottom of the side, which could be only achieved with computerised pipetting equipment. This would also eliminate timing differences between injecting 3-chloropropanediol in both hemispheres and ensure equal distribution of this neurotoxic agent. Also, cell counts on both pre-stained and individually stained slides were taken purely subjectively. Having each person to observe the full 28-day glial reaction would solve this problem and provide quantitative results on which statistical analysis could be conducted. Not all the groups recorded differences between the staining in medial and lateral inferior colliculus. Finally, there is the possibility of slides being cut at a slight angle, which would increase the volume of one hemisphere and lead to asymmetric cell distribution on hemispheres and error in cell counts.

Further analysis

Finally, further investigation of cellular response to brain injury could include quantitative analysis of protein density measured by densitometry analysis. Moreover analysis of post-transcriptional mRNA could be performed for specific markers such as Erg-1 and C1q, providing detailed analysis of the localised and generalised cellular reactions. Another additional study could involve comparison of different types of brain insults such direct injection with inflammatory factors (IL-1β or TNFα) into the brain regions. Gene knockouts and use of anti-inflammatory agents could further reveal the contribution of individual factors to the general response to injury.

Appendix 1. Figures and tables

Figure 1. Cell count and morphology of glial cells in rat inferior colliculus under the light microscopy after the treatment with 3-chloropropanediol.

Stain type

Appearance under x10 magnification Cell count and morphology

GFAP (4d)

Medial: +/- Lateral: ++ Cell shape: stellate

ED1 (6d)

Medial: +++ Lateral: +++ Cell shape: rounded

Table 2. Class scores for GFAP and ED1 staining in rat inferior colliculus at different times of survival.

Type of stain Survival time Cell count Morphology

0h

+/- -

3h +/- (M) + (L) +/-

6h +/- +/-

12h +/- +/-

18h +/- +/-

24h +/- +/-

2d

+/- +/-

3d

+/- (M) ++ (L) +/- (M) + (L)

4d +/- (M) + (L) - (M) ++ (L)

stellate

6d - n/a

8d +/- n/a

rounded

10d +/- n/a

14d +/- n/a +/-

GFAP

28d +/- +/-

stellate

0h

+ -

3h

+ +/-

6h ++ +

12h +/- +/-

18h + +

stellate

stellate/rounded

24h +/- +/-

2d ++ n/a

rounded

stellate/ rounded 3d ++ (M) +++ (L) ++ (M) +++ (L)

4d +++ ++

6d

+++ +++

8d +++ +++

10d +++ n/a

14d ++ (M) +++ (L) n/a

ED1

28d +++ +++

rounded

Cell counts key: n/a – class record missing; (-), absent; (+/-) less than the surrounding tissue; (+), little more than surrounding tissue; (++), more than the surrounding tissue; (+++) significantly more than the surrounding tissue

Figure 2. Results of GFAP staining using secondary antibody (a) and primary and secondary antibody (b-e) viewed under the light microscopy (x10)

b

a - artery (arachinoid space), b – ependyma (brain surface), c – artery (subarachidonic space), d – white matter, e – grey matter

References

Cavanagh, J.B, Nolan, C.C., (1993). The neurotoxicity of alpha-chlorohydrin in rats and mice: II. Lesion topography and factors in selective vulnerability in acute energy deprivation syndromes. Neuropathology and Applied Neurobiology. Vol. 19, pp.: 240-252

Dissing-Olesen, L., Ladeby, L., Nielsen, H.H., Toft-Hansen, H., Dalmau, I., Finsen, B. (2007). Axonal lesion-induced microglial proliferation and microglial cluster formation in the mouse. Neuroscience, Vol. 149 (1), pp.: 112–122

Fawcettt, J. W., Rosser, A. E., Dunnett, S. B. (2001) Brain damage, brain repair, Oxford University Press

Friedlander, R.M., Gagliardini, V., Rotello, R.J., Yuan, J. (1996) Functional role of interleukin 1 beta (IL-1 beta) in IL-1beta converting enzyme-mediated apoptosis, Journal of experimental Medicine Vol. 184, 717-724

Gehrmann, J., Matsumoto, Y., Kreutzberg, G.W. (1995). Microglia: intrinsic immuneffector cell of the brain. Brain Research Reviews, Vol. 20 (3), pp.: 269–287

Liedtke, W., Edelmann, W., Bieri, P.L., Chiu, F.C., Cowan, N.J., Kucherlapati, R., Raine, C.S. (1996) GFAP is necessary for the integrity of CNS white matter architecture and long-term maintenance of myelination. Neuron Vol. 17, pp.: 607-615

Ludwin, S.K., Kosek, J.C., End, L.F. (1976) The topographical distribution of S-100 and GFA proteins in the adult rat brain: an immunohistochemical study using horseradish peroxidase-labelled antibodies. Journal of Comparative Neurology Vol. 165, pp.: 197-207

Privat, A., Gimenez-Ribotta, M., Ridet, J.C. (1995). Morphology of astrocytes. In: Neuroglia (Ransom, B.R., Kettenmann, H., eds), New York: Oxford University Press, pp.: 3-22

Ritter, M.R., Banin, E., Moreno, S.K., Aguilar, E., Dorrel, M.I., Friedlander, M., (2006). Myeloid progenitors differentiate into microglia and promote vascular repair in a model of ischemic retinopathy. Journal of Clinical Investigation. Vol 116, (12), pp.: 3266–3276

Streita, W. (2006). Microglial senescence: does the brain's immune system have an expiration date?. Trends in Neurosciences Vol. 29 (9), pp.: 506–510

Tower, D.B., Young, O.M. (1973). The activities of butyrylcholinesterase and carbonic anhydrase, the rate of anaerobic glycolysis, and the question of constant density of glial cells in cerebral cortices of various mammalian species from mouse to whale. Joural of Neurochemistry Vol. 20, pp.: 269-278

Willis, C.L., Leach, L., Clarke, G.J., Nolan, C.C., Ray, D.E. 2004. Reversible disruption of tight junction complexes kn the rat blood brain barrier, following transitory focal astrocyte loss. Glia, Vol. 48, pp.: 1-13