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Microglial activation correlates with disease progression and upper motor neuron clinical symptoms in amyotrophic lateral sclerosis.

Brettschneider J, Toledo JB, Van Deerlin VM, Elman L, McCluskey L, Lee VM, Trojanowski JQ - PLoS ONE (2012)

Bottom Line: Microglial pathology as depicted by CD68 and Iba1 was significantly more extensive in the corticospinal tract (CST) of ALS cases with a rapid progression of disease.TDP-43 pathology was more extensive in the motor cortex of cases with rapid progression of disease.This study demonstrates that microglial pathology in the CST of ALS correlates with disease progression and is linked to severity of UMN deficits.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America. Johannes.brettschneider@uni-ulm.de

ABSTRACT

Background/aims: We evaluated clinicopathological correlates of upper motor neuron (UMN) damage in amyotrophic lateral sclerosis (ALS), and analyzed if the presence of the C9ORF72 repeat expansion was associated with alterations in microglial inflammatory activity.

Methods: Microglial pathology was assessed by IHC with 2 different antibodies (CD68, Iba1), myelin loss by Kluver-Barrera staining and myelin basic protein (MBP) IHC, and axonal loss by neurofilament protein (TA51) IHC, performed on 59 autopsy cases of ALS including 9 cases with C9ORF72 repeat expansion.

Results: Microglial pathology as depicted by CD68 and Iba1 was significantly more extensive in the corticospinal tract (CST) of ALS cases with a rapid progression of disease. Cases with C9ORF72 repeat expansion showed more extensive microglial pathology in the medulla and motor cortex which persisted after adjusting for disease duration in a logistic regression model. Higher scores on the clinical UMN scale correlated with increasing microglial pathology in the cervical CST. TDP-43 pathology was more extensive in the motor cortex of cases with rapid progression of disease.

Conclusions: This study demonstrates that microglial pathology in the CST of ALS correlates with disease progression and is linked to severity of UMN deficits.

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Related in: MedlinePlus

Staging of CST degeneration in ALS.The figure illustrates the IHC staging used to grade the extent of microglial activation (CD68, Iba1) and axonal loss (MBP, KB, NF) in the CST of ALS patients. Images are taken from the lateral portion of the cervical CST. KB  =  Kluver-Barrera, MBP =  myelin basic protein, NF  =  neurofilaments (TA51). Large images were taken with 4× objective, Scale bar is 1.0 mm. Small insert images were taken with 60× objective. Small insert image for CD68 stage “0” shows prominent neuronal nuclei, but no activated microglia.
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pone-0039216-g001: Staging of CST degeneration in ALS.The figure illustrates the IHC staging used to grade the extent of microglial activation (CD68, Iba1) and axonal loss (MBP, KB, NF) in the CST of ALS patients. Images are taken from the lateral portion of the cervical CST. KB  =  Kluver-Barrera, MBP =  myelin basic protein, NF  =  neurofilaments (TA51). Large images were taken with 4× objective, Scale bar is 1.0 mm. Small insert images were taken with 60× objective. Small insert image for CD68 stage “0” shows prominent neuronal nuclei, but no activated microglia.

Mentions: Sections of 6–10 µm thickness were cut from paraffin-embedded specimens. For IHC all slides were deparaffinized and rehydrated in a series of xylene and graded ethanol. After immersion in methanol/H2O2 for 30 min, slides were washed in 0.1 M Tris buffer (pH 7.6) and blocked in 0.1 M Tris/2% FBS. Sections were stained using polyclonal rabbit anti-Iba1 antibody (Wako Chemicals, Richmond, VA) at 1∶1.000 and incubated overnight at 4°C. Sections were then rinsed and washed in Tris and incubated with Vector biotinylated anti-rabbit IgG (Vector Laboratories Inc., Burlingame, Ca) at 1∶1.000 for 1 h. After rinsing again the ICH reaction was visualized using 3,3′-diaminobenzidine (DAB) and the sections were dehydrated through graded ethanol, cleared in xylene, and coverslipped in Cytoseal 60 mounting medium. Sections were stained for CD68 using mouse anti-human CD68 (Dako, Carpinteria, CA) at 1∶1.000. The extent of microglial activation was rated for each region on a 4-point ordinal scale (0, none; 1, mild; 2, moderate; 3, severe/numerous) as previously described [38], [39] (Figure 1). Staining for myelin basic protein (MBP) was performed as described before [40], as was Kluver-Barrera (KB) staining, while IHC for the phosphorylated species of the two neurofilament heavy chains (Nf) was used to assess axonal loss in the anterior and lateral CST of these ALS subjects. Staining of Nf was performed using TA51 which recognizes a phosphorylation-dependent epitope in the carboxy terminus of the high and middle molecular weight NF subunits as described [41], [42]. The primary antibody (supernatant) was used at 1∶200. This antibody is considered highly specific of Nf and does not cross-react with other cytoskeletal proteins [15].


Microglial activation correlates with disease progression and upper motor neuron clinical symptoms in amyotrophic lateral sclerosis.

Brettschneider J, Toledo JB, Van Deerlin VM, Elman L, McCluskey L, Lee VM, Trojanowski JQ - PLoS ONE (2012)

Staging of CST degeneration in ALS.The figure illustrates the IHC staging used to grade the extent of microglial activation (CD68, Iba1) and axonal loss (MBP, KB, NF) in the CST of ALS patients. Images are taken from the lateral portion of the cervical CST. KB  =  Kluver-Barrera, MBP =  myelin basic protein, NF  =  neurofilaments (TA51). Large images were taken with 4× objective, Scale bar is 1.0 mm. Small insert images were taken with 60× objective. Small insert image for CD68 stage “0” shows prominent neuronal nuclei, but no activated microglia.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375234&req=5

pone-0039216-g001: Staging of CST degeneration in ALS.The figure illustrates the IHC staging used to grade the extent of microglial activation (CD68, Iba1) and axonal loss (MBP, KB, NF) in the CST of ALS patients. Images are taken from the lateral portion of the cervical CST. KB  =  Kluver-Barrera, MBP =  myelin basic protein, NF  =  neurofilaments (TA51). Large images were taken with 4× objective, Scale bar is 1.0 mm. Small insert images were taken with 60× objective. Small insert image for CD68 stage “0” shows prominent neuronal nuclei, but no activated microglia.
Mentions: Sections of 6–10 µm thickness were cut from paraffin-embedded specimens. For IHC all slides were deparaffinized and rehydrated in a series of xylene and graded ethanol. After immersion in methanol/H2O2 for 30 min, slides were washed in 0.1 M Tris buffer (pH 7.6) and blocked in 0.1 M Tris/2% FBS. Sections were stained using polyclonal rabbit anti-Iba1 antibody (Wako Chemicals, Richmond, VA) at 1∶1.000 and incubated overnight at 4°C. Sections were then rinsed and washed in Tris and incubated with Vector biotinylated anti-rabbit IgG (Vector Laboratories Inc., Burlingame, Ca) at 1∶1.000 for 1 h. After rinsing again the ICH reaction was visualized using 3,3′-diaminobenzidine (DAB) and the sections were dehydrated through graded ethanol, cleared in xylene, and coverslipped in Cytoseal 60 mounting medium. Sections were stained for CD68 using mouse anti-human CD68 (Dako, Carpinteria, CA) at 1∶1.000. The extent of microglial activation was rated for each region on a 4-point ordinal scale (0, none; 1, mild; 2, moderate; 3, severe/numerous) as previously described [38], [39] (Figure 1). Staining for myelin basic protein (MBP) was performed as described before [40], as was Kluver-Barrera (KB) staining, while IHC for the phosphorylated species of the two neurofilament heavy chains (Nf) was used to assess axonal loss in the anterior and lateral CST of these ALS subjects. Staining of Nf was performed using TA51 which recognizes a phosphorylation-dependent epitope in the carboxy terminus of the high and middle molecular weight NF subunits as described [41], [42]. The primary antibody (supernatant) was used at 1∶200. This antibody is considered highly specific of Nf and does not cross-react with other cytoskeletal proteins [15].

Bottom Line: Microglial pathology as depicted by CD68 and Iba1 was significantly more extensive in the corticospinal tract (CST) of ALS cases with a rapid progression of disease.TDP-43 pathology was more extensive in the motor cortex of cases with rapid progression of disease.This study demonstrates that microglial pathology in the CST of ALS correlates with disease progression and is linked to severity of UMN deficits.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America. Johannes.brettschneider@uni-ulm.de

ABSTRACT

Background/aims: We evaluated clinicopathological correlates of upper motor neuron (UMN) damage in amyotrophic lateral sclerosis (ALS), and analyzed if the presence of the C9ORF72 repeat expansion was associated with alterations in microglial inflammatory activity.

Methods: Microglial pathology was assessed by IHC with 2 different antibodies (CD68, Iba1), myelin loss by Kluver-Barrera staining and myelin basic protein (MBP) IHC, and axonal loss by neurofilament protein (TA51) IHC, performed on 59 autopsy cases of ALS including 9 cases with C9ORF72 repeat expansion.

Results: Microglial pathology as depicted by CD68 and Iba1 was significantly more extensive in the corticospinal tract (CST) of ALS cases with a rapid progression of disease. Cases with C9ORF72 repeat expansion showed more extensive microglial pathology in the medulla and motor cortex which persisted after adjusting for disease duration in a logistic regression model. Higher scores on the clinical UMN scale correlated with increasing microglial pathology in the cervical CST. TDP-43 pathology was more extensive in the motor cortex of cases with rapid progression of disease.

Conclusions: This study demonstrates that microglial pathology in the CST of ALS correlates with disease progression and is linked to severity of UMN deficits.

Show MeSH
Related in: MedlinePlus