A must-see for TBI research! In the acute phase and 2 months later, the mIHC fluorescence polystaining index is more accurate

In the study of traumatic brain injury (TBI), the pathological process can change significantly over time. There are obvious differences in core pathological events at different stages, which makes the selection of multiple immunofluorescence staining (mIHC) indicators particularly critical. Today, let's talk about which indicators are recommended for mIHC fluorescence polystaining in the acute phase and the last two months of TBI.

 

1. Acute phase of TBI (several hours to 7 days after injury): focus on "injury initiation and early response"

 

Core pathologies in the acute phase include acute necrosis/apoptosis of neurons, blood-brain barrier (BBB) destruction, rapid activation of glial cells, and early inflammatory storm initiation. Therefore, mIHC indicators should focus on covering the four dimensions of cell survival and damage, glial activation, inflammation initiation, and BBB destruction.  

 

Recommended core indicators and significance

 

✦ Neuronal damage and survival: NeuN (neuronal nuclear antigen) can specifically label mature neurons and can be used to quantify neuronal loss or survival status, because neuronal necrosis/apoptosis in the acute phase will lead to a decrease in NeuN expression. Cleaved caspase-3 (activated caspase-3) is a marker of apoptotic cells, which can reflect the degree of apoptosis in acute phase. Co-staining with NeuN can also clarify the proportion of neuronal apoptosis.

 

✦ Glial cell activation: GFAP (glial fibrillary acidic protein) is a specific marker of astrocytes. In the acute phase, astrocytes will be rapidly activated, manifested as hypertrophy and hyperplasia, and the expression of GFAP will also be significantly up-regulated, which can reflect the glial stress response. Iba1 (ionized calcium binding adapter molecule 1) is a specific marker of microglia/macrophages. In the acute phase, microglia will quickly transform from the resting state to the activated state (amoeba-like). Iba1 can mark its quantity and morphological changes to reflect the initiation of inflammation.

 

✦ Inflammatory initiation: TNF-α (tumor necrosis factor-α) or IL-1β (interleukin-1β) are early pro-inflammatory factors secreted by activated microglia/astrocytes and are the core drivers of inflammatory storms. Co-staining with Iba1/GFAP can clarify the source of inflammation.

 

✦ Blood-brain barrier disruption: Claudin-5 (occludin-5) or ZO-1 (claudin-1) is the core component of BBB tight junctions. BBB disruption in the acute phase will reduce its expression or disorder its distribution. Combined with vascular markers (such as CD31) can assess BBB integrity.

 

Recommended combination (4-6 colors)    

✦ Basal combination: NeuN (neurons) + GFAP (astrocytes) + Iba1 (microglia) + Cleaved caspase-3 (apoptosis)

✦ Extended combination (with BBB/inflammation): NeuN + GFAP + Iba1 + Claudin-5 + TNF-α

 

2. 2 months after TBI (chronic phase): focus on "repair and long-term pathology"

 

It enters the chronic stage 2 months after injury, and the core pathologies include chronic neuroinflammation, glial scar formation, nerve regeneration attempt, and axonal/synaptic remodeling disorder. At this time, mIHC indicators should focus on covering the four dimensions of chronic glial activation, nerve regeneration, structural remodeling, and inflammatory phenotype conversion.

 

Recommended core indicators and significance   

 

1) Neuronal regeneration and survival: NeuN still needs to be used to monitor the long-term survival status of mature neurons, because neuronal loss in chronic phase may persist. DCX (dicortin) can specifically label newborn neurons (immature neurons), which can reflect endogenous neurogenesis ability. After all, the subependymal area may initiate nerve regeneration after TBI.

 

2) Chronic glial activation and scar formation: In the chronic phase of GFAP, astrocytes will continue to activate and participate in glial scar formation, and the shape is denser. Combined with its distribution, the scar scope can be evaluated. Vimentin (Vimentin) is co-expressed with GFAP in chronically activated astrocytes, which can enhance the marking of glial scars, because the expression of Vimentin in astrocytes in the scar area is upregulated.

 

3) Microglia inflammatory phenotypic switch: Iba1 can still mark microglia, but it should be combined with phenotypic markers to distinguish functional status. CD16/32 is a marker of pro-inflammatory phenotype (M1 type), which can reflect chronic pro-inflammatory state, which may aggravate nerve damage; CD206 is an anti-inflammatory/repair phenotype (M2 type) marker, which can reflect neuroprotective tendencies and is conducive to tissue repair.

 

4) Axon and synaptic remodeling: NF-H (neurofilament heavy chain) or β-III tubulin can label axons and be used to evaluate degeneration after chronic axon regeneration or rupture (such as Wallerian degeneration). PSD-95 (Postsynaptic Compact-95) or Synaptophysin (Synaptic Vesicle Protein) label the postsynaptic membrane and synaptic vesicles respectively, which can reflect the number and functional status of synapses. It is necessary to know that synaptic remodeling disorder in chronic phase is the key to poor recovery of neurological function.

 

5) Angiogenesis (optional): CD31 (platelet endothelial cell adhesion molecule) can label vascular endothelial cells and be used to evaluate brain angiogenesis in chronic phase, which is related to blood supply support for nerve repair.

 

Recommended combination (4-6 colors)

✦ Basic combination: NeuN (mature neurons) + DCX (newborn neurons) + GFAP (astrocytes) + Iba1 + CD206 (anti-inflammatory microglia)

✦ Extended combinations (including synaptic remodeling ): NeuN + DCX + GFAP + Iba1 + PSD-95 + NF-H

 

3. Selection precautions

 

1) Antibody compatibility: mIHC does not need to avoid antibody species conflicts. For example, primary antibodies can be of the same species and are both rabbit antibodies. It is recommended to give priority to monoclonal primary antibodies.

 

2) Fluorescein matching: To avoid the overlap of fluorescence spectra, if 8-10 colors are to be made, the signal can be optimized by spectral resolution technology.

 

3) The research focuses on adjustment: if you focus on "oxidative stress", 4-HNE (4-hydroxynonenal) can be added in the acute phase; If you are concerned about "iron deposition", Ferritin (Ferritin) can be added in the chronic phase.

 

Through targeted selection of indicators, the pathological characteristics of different stages of TBI can be clearly revealed, providing visual evidence for mechanism research (such as inflammatory regulation, nerve regeneration) or intervention targets (such as regulating microglia phenotype and promoting synaptic remodeling). I hope this content can be helpful to you who are engaged in TBI research!

 

mIHC Select Absin:

1) Optional 1-10 colors;

2) Leica automatic staining, immediately staining panel package antibody;

3) Package scanning (3D & TG scanner);

4) Package analysis (HALO analysis software);

5) 7-10 colors complimentary hard disk + organizational streaming scatter analysis;

6) Results will be available in 1 week at the fastest.

 

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. Email: worldwide@absin.cn

Follow us on Facebook: Absin Bio