Zebrafish And Pro-inflammatory Cytokine
During spinal cord injury, prolonged inflammation in the injury site Makes Re-generation of nerve cell connections difficult. So in order to understand in detail the responsible factor that causes inflammation we use zebrafish as our model organism which uses blood-derived macrophage for recovery from spinal cord injury.
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We designed an in vivo experiment for screening of immune system in larval zebrafish with help of CRISPR CAS9 Technology in order to study how larval zebrafish use blood-derived macrophage which keeps neutrophils and il1 b – pro-inflammatory cytokine expression up to date during recovery of spinal cord injury. We found “4 genes – thfb 1a, tgfb 3, tnfa, sparc ” of sCrRNAs are highly active and showed successful regeneration of spinal cord in larval zebrafish. Finally using a CRISPR-based phenotypic screen in larval zebrafish thfb 1 gene appears to play an essential role in controlling inflammation during the recovery process.
Spinal Cord Injuries (SCI) And Pro-inflammatory Cytokine Involved During Spinal Cord Regeneration:
- Inflammation after SCI is complex and orchestrated by many cell types and numerous inflammatory cytokines including tumor necrosis factor-alpha (TNFα), interleukin-1β (IL-1β), and interleukin-6 (IL-6).
- However in general key pro-inflammatory cytokines are IL-1, IL-6, and TNF-α. These cytokines signal via type I cytokine receptors (CCR1) that are structurally divergent from other cytokine receptor types. They are crucial for coordinating cell-mediated immune response and play a critical role in modulating the immune system.
Why Zebrafish as a Model?
- Zebrafish can regenerate axonal connections on spinal cord injury sites. Prolonged inflammation causes harm and prevents recovery during spinal injury in mammals, but in zebrafish, pro-inflammatory cytokines are rapidly down-regulated and the immune response generally promotes regeneration.
- Readily available genetic information, fast and reliable manipulation of genes and its fecundity also makes zebrafish a perfect model for this study.
CRISPR CAS 9?
CRISPR –clustered regularly interspaced short palindromic repeats
- DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea.
- Sequences derived from DNA fragments of bacteriophages which previously infected the prokaryote.
- Used to detect and destroy DNA from similar bacteriophages during subsequent infections.
- Hence these sequences play a key role in the antiviral (anti-phage) defence system of prokaryotes and provide a form of acquired immunity.
Cas 9-CRISPR-associated protein 9
- It’s an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence.
- Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms.
Modeling Spinal Cord Injury With CRISPR CAS 9?
sCrRNAs, TracrRNA, Cas9 restriction enzymes were taken. sCrRNAs are designed in a way that the restriction enzyme recognition sequences can overlap the Cas9 cut site. For in vivo testing a mix of sCrRNAs, TracrRNA, Cas9 enzymes (CrRNAs targeting exon 1), and fast green dye each at 1 μl ratio is injected into the yolk of single-stage cell embryos. For generating stable mutants CrRNAs targeting sites tgfb 1a, tgfb 3 were taken, tnfa, sparc, cst 7 were targeted with respective strands of sCrRNAs to get a larger deletion. Our desired traits are identified and crossed with wild-type genes. All spinal cord lesion assays were performed on an F3 heterozygous incross.
Parameters To Analyse Which Pro-inflammatory Cytokine is Responsible for Spinal Cord Injury Regeneration:
Restriction fragment length polymorphisms (RFLP), Allele sequencing, Hexb activity assay, Immunohistochemistry on whole-mount larvae. After we have done spinal cord injury and compound incubation. At last, we have done behavioral assay and at last RT-PCR.
With the help of already available information in cytokines involved in inflammation we focused on tgfb 1a and tgfb3 . We found that tgfb1a as a regulator of post-injury inflammation helps us to understand how inflammation is rapidly resolved to promote recovery. Thus we conclude that immune reaction is essential for spinal cord regeneration. This discovery may help researchers to invent drug compounds to cure rare neurodegenerative disease, also on enhancing recovery state of spinal cord injury patients.