Thyroxin regulates BDNF expression to promote survival of injured neurons

Done by: Sumaiah Alghamdi434203445

Submitted to Dr : Afrah Al khorji2015

Objective

• Introduction• Study • material and methods• Result• Discussion• Summary

Introduction • thyroid hormones work on proper fetal

growth and development, particularly development of the central nervous system (CNS).

• regulation of energy metabolism.• During development, thyroid hormone

levels are critical for the growth of cerebral and cerebellar cortex

• many studies have shown that thyroid hormones promote the regeneration of peripheral nerves after axotomy

Introduction cont..

Study

Study ..cont

• In this study show that thyroid hormones promote maturation of oligodendrocytes and remyelination after traum

• Data showing the trophic effect of thyroid hormones on axotomized CNS neurons are scarce and contradictory This prompts for further investigation on the therapeutic potential of this hormone.

• In humans have shown that during the first few days after traumatic brain injury or spinal cord injury, thyroid hormone levels are often diminished.

Study ..contMany studies have reported analogies between

developmental and post-traumatic events in the central nervous system:

1- BDNF (brain-derived neurotrophic factor.)

The injured neurons

dependency on BDNF

the neurons lost the dependency

on BDNF

the neurons cell dependency on

BDNF

after trauma during maturation

in developing neurons

Study ..cont

2- GABAA-mediated receptor ( is an ionotropic receptor and ligand-gated ion channel)

Depolarizing Hyper polarizing

Depolarizing

after trauma during maturation

in developing neurons

Study ..cont

3- GABAa changes reflect the variation in the functional levels of K-Cl cotransporter (KCC2) ( Potassium-chloride transporter) KCC2 expression is required for hyper polarizing GABAA responses during maturation of neuron .

• Our data suggest that maintaining normal levels of thyroxin during the early post-traumatic phase of CNS injury could have a therapeutically positive effect.

Material & methods

1- Hippocampal slices (The hippocampus is located in the medial temporal lobe of the brain)

Hippocampal slices from 8- to 9-day-old male and female rats.

Material & methods• First Region of hippocampus proper (CA1) is a

portion of the hippocampal formation. neurons cell bodies are in CA3 and whose axons project to CA1. however, it contains very few recurrent connections. In the rat, CA1 contains approximately 250,000 pyramidal cells

• The second Region the dentate gyrus (DG), is actually a separate structure, a tightly packed layer of small granule cells.

Material & methods

2-Lesion procedureSelected slices, were lesioned between CA3 and CA1

regions using a razor blade.

Material & methods

3- Real-time PCR4- 3D image analysis Images stacks consisting of 11

images from 10 and 20 μm5-Anterograde axonal labeling Organotypic The dye

Alexa658 was injected into CA3 region for anterograde labeling for collateral axons.

6-Immunohistochemical staining was performed for nuclear staining (-NeuN was used to estimate neuronal number ,Hoechst was used to estimate the effect in the total number of cells and cleaved caspase-3 which is a critical executioner of apoptosis)

Material & methods

7- Microfluidic culture platform and time-lapse microscopy

This method allows the specific isolation of axons of primary neuronal cultures.

8- StatisticsThe data are presented Statistical analysis was

performed using non-paired test on MS Excel software.

Result 1-The effect of thyroxin on BDNF mRNA expression levels

changes after injury from down-regulation to up-regulation

Lesioned slices treated with 1-μMthyroxin had 25% higher BDNF mRNA levels than lesioned control slices. In unlesioned slices treated with thyroxin, BDNF mRNA levels were 15% lower than in unlesioned control slices

Result (cont .. )

• These data indicate that down-regulation of BDNF mRNA by thyroxin is changed to a dose-dependent upregulation after trauma.

Result (cont .. )

2-Thyroxin promotes survival of damaged neurons in a BDNF-dependent manner

• NeuN staining ,Hoechst staining and cleaved caspase-3 staining been used.

Result (cont .. )

Lesioned slices treated with 10 μM thyroxin , 0.1 μM and 0.01 μM are normalized to lesioned controls

Result (cont .. )

3- Thyroxin promotes regeneration of injured neurons BDA-conjugated Alexa 658 was electroporated into CA3for anterograde labeling of Schaffer collateral axons.the dye could be seen passing across the cut area to the

CA1 region. • In unlesioned control slices, accumulation of the dye

in CA1 region was seen in 100% of slices• In lesioned control slices the dye was able to pass

over the lesioned area in 25% of slices.• In lesioned slices treated with thyroxin the dye was

seen in CA1 region in 100% of slices

Result • (A,D) unlesioned; (B,E) lesioned; (C,F ) lesioned

treated with thyroxin

Result 4-Thyroxin up-regulates KCC2 in lesioned and down-

regulates KCC2 in unlesioned neuronsneurons that survive after axotomy gradually regain higher K-

Cl cotransporter (KCC2) expression levels

Real-time PCR analysis Thyroxin up-regulated KCC2 mRNA levels by 47% in lesioned slices and down-regulated KCC2 Mrna by 54% in unlesioned slices

Discussion

• Previous results have shown that thyroxin levels are diminished after acute spinal cord injury or traumatic brain injury .

• Thyroxin would elevate BDNF expression, promoting survival of neurons, and in parallel might be also beneficial for the survival of the glial cells .

• Our results indeed indicate that thyroxine promotes survival and regeneration of neurons after axotomy in a BDNF-dependent manner. thyroxine can up-regulate KCC2 in traumatized neurons. promoting Their “re-maturation” and restoring normal functioning of GABAergic inhibition.

Discussion

• These results prompt the question whether thyroxin could be used as a therapeutic agent after trauma. ??

Summary • In summary, we have shown that a switch can take

place at a higher level of cell signaling,1) at the level of hormone–neurotrophin interaction

(thyroxine–BDNF) 2) as well as in hormone–protein

regulation(thyroxine–KCC2). • Deeper understanding of the mechanism of the

“developmental switch” of injured CNS could lead to new strategies of therapeutic importance that would use the supply of a developmental environment for injured neurons during the acute post-traumatic period as a basic strategy.

Any Question??