Human platelet lysate, a novel and smart thoroughfare for the treatment of CNS disorders

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TBI burden on patients, their family, and the community

As it can be seen through the incidence worldwide, TBI is devastating the lives of millions of people. For example, the severe TBI mortality rate is estimated to 30-40%22. The disease burden affects life conditions at the individual, family, and community levels. Patients who survived the injury could experience physical disability, psychiatric, cognitive, and emotional impairments. The long-term impact includes memory loss, decreasing abilities in managing stress, and temper. These situations affect the patient’s relationships and, finally, their life in society.
TBI patients’ management requires meticulous care and is everlasting. Clinicians and patients’ families are always at the front line in this battle. Unfortunately, this uncomfortable situation also required the participation of the community. According to previously published data, approximately $400 billion is spent annually worldwide for TBI patients’ management8. This colossal amount highlights the urgency and the implication of researchers, policymakers, therapeutists, and patient families at all levels in order to offer better living conditions to TBI patients.

Traumatic brain injury classes

The brain injury’s heterogeneity and complexity led to TBI stratification according to the clinical severity, the injury mechanism, and pathophysiology.

Traditional classification method of head injuries

The traditional classification of TBI is based on the type of injury and its severity, and four categories have been defined as described by Pushkarna et al., (2010)23:
● Open injuries: this category of injury commonly happens in combat.
● Closed injuries: are predominantly seen in civilian and military operations.
● Scalp injuries: this type of injury can be closed or open.
● Skull fractures: a group of cases with a skull fracture.
In addition to the traditional classification, another standard scale is used during the clinical management of brain injury, the Glasgow Coma Scale (GCS). Three categories of TBI is defined based on this scale: mild (scores ≥ 13), moderate (scores 9–12), and severe (scores 3–8) and some physicians determine TBI outcomes with the GCS outcome scale, which estimates the neurobehavioral ranges of recovery such as: dead; vegetative state; severe or moderate impairment; and well recovery24,25 (see Table 1).

Classification using neuroimaging scales

Imaging instruments such as computer tomography (CT) and magnetic resonance imaging (MRI), developed for TBI diagnosis and management, can locate, or identify a skull fracture, intracranial hemorrhage, epidural and subdural hematoma, subarachnoid hemorrhage, intra-parenchymal hemorrhage, intraventricular hemorrhage, focal and diffuse patterns of axonal injury with cerebral edema, and cerebral contusion26. The use of these imaging systems allowed us to define the Marshall and the Rotterdam scales. The Marshall scale uses CT imaging to define six types of injuries, as summarized in Table 2. In order to pave the limitations of the Marshall scale, the Rotterdam scale was proposed. However, this promising tool must undergo further validation steps.

Table of contents :

Table of content
List of figures
List of tables
List of abbreviations
ABSTRACT
Résumé en Français
Résumé substantiel en Français
ENGLISH ABSTRACT
CHAPTER I: General introduction
INTRODUCTION
1.1 Traumatic brain injury
1.2. Causes and epidemiology of traumatic brain injury
1.3 TBI burden on patients, their family, and the community
1.4 Traumatic brain injury classes
1.5 Physiopathology of TBI-related brain injury
1.6 TBI diagnosis tools
1.7 Experimental models of TBI
1.8 Current interventions
1.9 TBI and blood transfusions
1.10 Pharmacological drugs for TBI
1.11 Therapeutic strategies for neuronal recovery and neurobehavioral improvement
1.12 Neuropsychological Rehabilitation (NR) and Neurotherapy
1.13 New therapeutic strategies
2.1 Platelet production, structure, and physiology
2.2 Platelets function
2.3 Platelet concentrates
2.4 Human platelet lysates
3. Human platelet lysate, a novel and smart thoroughfare for the treatment of CNS disorders?
3.1 Platelet and brain function
3.2 Role of human platelet lysate in the modulation of neural stem cells (NSCs) proliferation, survival, and differentiation
3.3 Current investigation of the neuroprotective roles of human platelet lysate in CNS injury
4. Statement of aims
5. Aims
Chapter II: Materials and Methods
A. Overall study design
B. In vitro investigation of human platelet pellet lysate bioactivity
1. Human platelet lysate used in this study
1.1 The platelet concentrates
1.2 Preparation of the Heat-treated Platelet Pellet Lysates (HPPL)
1.3 Platelet lysate characterization
2. Cell cultures
2.1 LUHMES cell culture
2.2 SH-SY5Y cell maintenance
2.3 BV-2 microglia
2.4 Human Endothelial Cell Line (EA-hy926)
2.5 Primary cortical neuronal cultures
3. Safety and Functional assessment of HPPL
3.1 Safety assessment of HPPL and I-HPPL
3.2 Protective activity of HPPL/I-HPPL in a cell model of ferroptosis
3.3 In vitro scratch healing assay
3.4 Neurites outgrowth assay
C. In vivo study
1. Study approval and animal experiment design
2. Traumatic brain injury models and HPPL administration
2.1 Anesthesia and surgical preparation
2.2 Cortical brain scratch assay model
2.3 Mild traumatic brain injury model using the controlled cortical impact device
2.4 Treatment administration procedure
3. Animals behavior tests
3.1 Beam test
3.2 Rotarod test
3.3 Open field test
3.4 Novel object recognition (NOR) test.
4. Tissue collection and processing
5. Oxidative stress (ROS) detection
6. Western Blot analysis for synaptic proteins determination
7. ELISA for synaptic proteins assessment
8.1 RNA purification
8.2 RT-qPCR for inflammatory markers and oxidative stress
9. Immunofluorescence for histological analysis
9.1 Animal perfusion and brain samples handling procedure
9.2 Tissue sectioning and staining procedure
10. Proteomics analysis of mice cortex samples
11. Bioinformatics analysis.
Chapter III: Results
1. Heat-treated platelet pellet lysate is a biomaterial full of bioactive substances
2. Safety assessment of the heat-treated human platelet pellet lysate fractions
2.1 Impact of heat-treated platelet pellet lysates on cell viability
2.2 Impact of heat-treated platelet pellet lysates on SH-SY5Y cells membrane integrity .
2.3 Impact of heat-treated platelet pellet lysates on proteins expression
2.4 Impact of heat-treated human platelet pellet lysates on lipid peroxidation
2.5. HPPL and I-HPPL did not activate microglia BV-2 cells.
3. Investigation of heat-treated platelet lysates activity in vitro
3.1 Platelet lysates can enhance a wound healing
3.3 Anti-ferroptosis potential of heat-treated platelet pellet lysate fractions
B. In vivo neuroprotective effect of heat-treated platelet pellet lysate
1. Pathophysiological development of brain lesions in cortical brain scratch and CCI models
1.1 The brain scratch induced behavioral deficits in motor dependent tasks but not in novel object recognition at DPI7.
1.2 The scratch injury induced oxidative stress
1.3 The impact of the cortical brain injury on synapses
2. The functional outcomes of HPPL administration in TBI-mice
2.1 HPPL administration improved motor and cognitive function of TBI mice.
2.3 HPPL enhanced the expression of synaptic proteins in the cortex of mice
2.4 HPPL administration reduced a level of oxidative stress in the cortex of TBI mice.
3. What proteomics analysis told us about HPPL potential in brain injury models?
Chapter IV: Discussion
CONCLUSION
References