TRAJECTORY CLUSTERING USING LATENT CLASS MODELS FOR UNSUPERVISED TBI BIOMARKER TEMPORAL PHENOTYPE DISCOVER

Background: TBI biomarkers display population-level time-varying kinetics [1] which may be a rich source of pathobiological information [2]. At an individual level, deviations from stereotypical trajectories may represent different pathological processes or secondary insults. A method for discovering such phenotypes may be useful in in- dividualising treatments in real-time. Methods: Serial blood (12hourly) and CSF (6hourly) samples were obtained from seventeen adult patients with severe TBI (Stockholm ethics committee approval #2009/1112-31). S100B and neuron-specific enolase (NSE) concentrations were measured along with blood:CSF albumin quotient Qa as a measure of blood-brain-barrier (BBB) integrity. S100B and NSE concentrations were log-transformed: Equivalent to the assumption of baseline exponential decay. We used trajectory modeling combining a quadratic mixed effects model with latent group analysis to search for characteristic trajectories in the measured parameter. Results: For serum S100B, we discovered two phenotypes with fast and slow kinetics. The fast group corresponded with patients with more severe extracranial injury. For serum NSE, again two phenotypes were discovered; a time-decaying group and another with a peak around day 4. CSF analysis yielded two latent groups for both S100B and NSE: a time-decaying group and another displaying prolonged elevation over several days. Qa data clustered into three groups: two with fast, slow decay and another with prolonged elevation. The group with prolonged BBB permeability had corresponding poorer outcomes. Conclusions: Small numbers prevent statistical comparison, but trajectory modeling identified a number of phenotypes with plausible pathobiological significance. In particular the technique revealed a group of patients with secondary serum NSE release and another with sustained BBB permeability. Such groups seem to relate to injury profile and outcome suggesting biological relevance. To our knowledge this is the first use of an unsupervised clustering technique in kinetic phenotype discovery. References: [1] Ercole A, Thelin EP, Holst A, Bellander BM, Nelson DW. Kinetic modelling of serum S100b after traumatic brain injury. BMC Neurol. 2016;16:93. [2] Thelin EP, Zeiler FA, Ercole A, Mondello S, Büki A, Bellander BM, Helmy A, Menon DK, Nelson DW. Serial Sampling of Serum Protein Biomarkers for Monitoring Human Traumatic Brain Injury Dynamics: A Systematic Review. Front Neurol. 2017;8:300

Inhalation of Molecular Hydrogen, a Rescue Treatment for Noise-Induced Hearing Loss

Noise exposure is the most important external factor causing acquired hearing loss in humans, and it is strongly associated with the production of reactive oxygen species (ROS) in the cochlea. Several studies reported that the administration of various compounds with antioxidant effects can treat oxidative stress-induced hearing loss. However, traditional systemic drug administration to the human inner ear is problematic and has not been successful in a clinical setting. Thus, there is an urgent need to develop rescue treatment for patients with acute acoustic injuries. Hydrogen gas has antioxidant effects, rapid distribution, and distributes systemically after inhalation.The purpose of this study was to determine the protective efficacy of a single dose of molecular hydrogen (H-2) on cochlear structures. Guinea pigs were divided into six groups and sacrificed immediately after or at 1 or 2 weeks. The animals were exposed to broadband noise for 2 h directly followed by 1-h inhalation of 2% H-2 or room air. Electrophysiological hearing thresholds using frequency-specific auditory brainstem response (ABR) were measured prior to noise exposure and before sacrifice. ABR thresholds were significantly lower in H-2-treated animals at 2 weeks after exposure, with significant preservation of outer hair cells in the entire cochlea. Quantification of synaptophysin immunoreactivity revealed that H-2 inhalation protected the cochlear inner hair cell synaptic structures containing synaptophysin. The inflammatory response was greater in the stria vascularis, showing increased Iba1 due to H-2 inhalation.Repeated administration of H-2 inhalation may further improve the therapeutic effect. This animal model does not reproduce conditions in humans, highlighting the need for additional real-life studies in humans

Validation of Local Brain Kinematics of a Novel Rat Brain Finite Element Model under Rotational Acceleration

Relative brain-skull displacement under head rotational acceleration in rats was evaluated experimentally. For this, a thin pin was entered the cortex and rigidly attached to the skull prior to impact. For peak rotational accelerations of 1.7 Mrad/s2, the pin scarred the brain cortex; 1.2 mm superficially and less centrally. These measurements were used to validate the brain kinematics of a new anatomically detailed FE model of the head-neck complex of a rat. This model is intended to be used to clarify brain loading mechanisms and to develop brain tissue injury threshold for Diffuse Axonal Injuries as detected through immune-histology

Hippocampal Expression of Cytochrome P450 1B1 in Penetrating Traumatic Brain Injury

Hippocampal dysfunction contributes to multiple traumatic brain injury sequala. Female rodents' outcome is superior to male which has been ascribed the neuroprotective sex hormones 17 beta-estradiol and progesterone. Cytochrome P450 1B1 (CYP1B1) is an oxidative enzyme influencing the neuroinflammatory response by creating inflammatory mediators and metabolizing neuroprotective 17 beta-estradiol and progesterone. In this study, we aimed to describe hippocampal CYP1B1 mRNA expression, protein presence of CYP1B1 and its key redox partner Cytochrome P450 reductase (CPR) in both sexes, as well as the effect of penetrating traumatic brain injury (pTBI). A total 64 adult Sprague Dawley rats divided by sex received pTBI or sham-surgery and were assigned survival times of 1-, 3-, 5- or 7 days. CYP1B1 mRNA was quantified using in-situ hybridization and immunohistochemistry performed to verify protein colocalization. CYP1B1 mRNA expression was present in all subregions but greatest in CA2 irrespective of sex, survival time or intervention. At 3-, 5- and 7 days post-injury, expression in CA2 was reduced in male rats subjected to pTBI compared to sham-surgery. Females subjected to pTBI instead exhibited increased expression in all CA subregions 3 days post-injury, the only time point expression in CA2 was greater in females than in males. Immunohistochemical analysis confirmed neuronal CYP1B1 protein in all hippocampal subregions, while CPR was limited to CA1 and CA2. CYP1B1 mRNA is constitutively expressed in both sexes. In response to pTBI, females displayed a more urgent but brief regulatory response than males. This indicates there may be sex-dependent differences in CYP1B1 activity, possibly influencing inflammation and neuroprotection in pTBI

Observations at the CNS-PNS border of ventral roots connected to a neuroma

Previous studies have shown that numerous sprouts originating from a neuroma, after nerve injury in neonatal animals, can invade spinal nerve roots. In this study the border between the central and peripheral nervous system (CNS-PNS border) of ventral roots in kittens was examined with both light and electron microscopy after early postnatal sciatic nerve resection. A transient ingrowth of substance P positive axons was observed into the CNS, but no spouts remained 6 weeks after the injury. Using serial sections and electron microscopy it was possible to identify small bundles of unmyelinated axons that penetrated from the root fascicles for a short distance into the CNS. These axons ended blindly, sometimes with a growth cone-like terminal swelling filled with vesicles. The axon bundles were accompanied by p75 positive cells in both the root fascicles and the pia mater, but not in the CNS. It may thus be suggested that neurotrophin presenting p75 positive cells could facilitate axonal growth into the pia mater and that the lack of such cells in the CNS compartment might contribute to the failure of growth into the CNS. A maldevelopment of myelin sheaths at the CNS-PNS border of motor axons was observed and it seems possible that this could have consequences for the propagation of action potential across this region after neonatal nerve injury

Cellular High-Energy Cavitation Trauma - Description of a Novel In Vitro Trauma Model in Three Different Cell Types

The mechanisms involved in traumatic brain injury have yet to be fully characterized. One mechanism that, especially in high-energy trauma, could be of importance is cavitation. Cavitation can be described as a process of vaporization, bubble generation, and bubble implosion as a result of a decrease and subsequent increase in pressure. Cavitation as an injury mechanism is difficult to visualize and model due to its short duration and limited spatial distribution. One strategy to analyze the cellular response of cavitation is to employ suitable in vitro models. The flyer-plate model is an in vitro high-energy trauma model that includes cavitation as a trauma mechanism. A copper fragment is accelerated by means of a laser, hits the bottom of a cell culture well causing cavitation, and shock waves inside the well and cell medium. We have found the flyer-plate model to be efficient, reproducible, and easy to control. In this study, we have used the model to analyze the cellular response to microcavitation in SH-SY5Y neuroblastoma, Caco-2, and C6 glioma cell lines. Mitotic activity in neuroblastoma and glioma was investigated with BrdU staining, and cell numbers were calculated using automated time-lapse imaging. We found variations between cell types and between different zones surrounding the lesion with these methods. It was also shown that the injured cell cultures released S-100B in a dose-dependent manner. Using gene expression microarray, a number of gene families of potential interest were found to be strongly, but differently regulated in neuroblastoma and glioma at 24 h post trauma. The data from the gene expression arrays may be used to identify new candidates for biomarkers in cavitation trauma. We conclude that our model is useful for studies of trauma in vitro and that it could be applied in future treatment studies