Hiromichi Nakadate

Combinations of hydrostatic pressure and shear stress influence morphology and adhesion molecules in cultured endothelial cells

Endothelial cells are exposed to mechanical stimuli from blood flow and blood pressure. However, it is not yet fully understood how their simultaneous exposure affects endothelial function. Firstly, in this study we investigated the effect of combined stress on morphology of cultured human aortic endothelial cells (HAECs). In the results, HAECs exposed to steady flow (a pressure of 100 mmHg, and a shear stress of 1.5 Pa) were more elongated than those exposed to a hydrostatic pressure of 100 mmHg and HAECs exposed to pulsatile flow (a pressure of 80/120 mmHg, and a shear stress of 1.2/1.8 Pa) were more elongated than those exposed to steady flow. Similarly, HAECs exposed to pulsatile flow were most oriented to the flow direction among these three stresses. Secondly, we investigated the effect of combined stress on gene expression of cell adhesion molecules in HAECs. After stress exposure to HAECs the mRNA of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin were measured by real time RT-PCR. In the results, the exposure of steady flow increased the mRNA levels of ICAM-1 compared to the exposure of hydrostatic pressure; however, the exposure of pulsatile flow decreased the mRNA level of ICAM-1 compared to the exposure of steady flow. These findings suggest that gene expression of cell adhesion molecules induced by combined stress were different to the superposition of individual stress and that not only difference in the components of combined stress but also difference in the magnitude of the components of combined stress are important.

Stretch-induced functional disorder of axonal transport in the cultured rat cortex neuron

Background: Diffuse axonal injury (DAI) is a one of the main causes of traumatic brain injury and caused by the impulsive stretching of neuronal axons resulting from rapid deformation of a brain. DAI is characterized by a gradual swelling of the axons which is formed by the accumulation of cellular organelles and proteins, and the swelling is the morphological hallmark of DAI pathology. In recent years, the details of the stress and strain of the whole damaged brain are becoming clear with the rapid development of the computational mechanics and finite element (FE) head models are able to give better prediction to the brain injury and to evaluate the protective safety methods with more detailed neuronal tolerance criteria. In this study, axonal injury induced by precisely controlled impulsive strain and strain rate was evaluated and the tolerance criteria for the functional disorder for each dysfunction and disruption level of axonal transport was obtained by observation of β-amyloid precursor protein (β-APP) in cultured rat cortex neuron. Methods: The uniaxial stretching device which could give various combinations of strains and strain rates to neurons was developed. The various loading conditions for neurons were verified by comparing the experimental displacement history of the substrate taken by microscopy with the FE strain distribution analysis of culturing substrate. The primary rat cortex neurons were stretched by different combinations of strains and strain rates and β-APP was immunostained at 3h after loading and observed by fluorescence microscopy. Results: The number of swellings and bulbs formed on axons by β-APP-accumulation after stretching were observed and counted by fluorescent images. The dysfunction of the axonal transport was defined as the rate of neurons that have β-APP-accumulating axonal swellings and disruption of the axonal transport was defined as the rate of neurons that have β-APP-accumulating axonal bulbs, respectively. The degree of the functional disorder of the axonal transport advanced with the increase of strain and strain rate. Conclusions: The mechanical threshold of dysfunction and disruption of axonal transport were the strain with 0.22 and the strain rate with 27 /s. The intervals between swellings on an axon are constant and do not depend on the axonal injury level nor the magnitude of the strain of the axons.

Study on the Mechanism of Cerebral Contusion Based on Judicial Autopsy Report

Cerebral contusion is one of the symptoms observed when a human receives a heavy impact on the head, and considered to be caused by the negative pressure fluctuation inside a cranium. In order to know the mechanism of cerebral contusion, the impact experiment and finite element analysis of a water-filled acrylic container was carried out. In the analysis, mass and velocity of the impactor were changed, so as to keep the energy constant and intracranial pressure fluctuation inside the container was obtained. The frequency analyses were performed for the pressure fluctuations. It was shown that the higher frequency was excited and the negative pressure occurred at the impact side when the force duration was short, and consequently coup contusion was caused. 11 autopsied cases which had in-depth data were reconstructed by using finite element model of a human head. The autopsy reports included the data of the impact object, impact region, skull fracture region and lesion area of a brain. In analysis, the impact velocity was estimated so that the negative pressure could occur only at the lesion area of a brain. As a result of analysis, the force duration was short when the impact object was light weight with high velocity, the negative pressure occurred at the impact side and the positive pressure occurred at the opposite side of impact, and consequently coup contusion was obtained. the force duration was long when the impact object was heavy weight with low velocity, the positive pressure occurred at the impact side and the negative pressure occurred at the opposite side of impact, and consequently contrecoup contusion was obtained.

Impact Pressure Increases Permeability of Cultured Human Endothelial Cells

Traumatic brain injury (TBI) is well known to trigger multiple brain parenchymal and vascular responses. The immediate and prolonged opening of blood-brain barrier (BBB) is a hallmark of TBI pathophysiology, and results in extravasation of blood components, including red blood cells, plasma proteins and water (vasogenic edema) [1]. On the other hand, Studies in impact biomechanics have demonstrated a number of brain injury mechanisms [2]. These mechanisms include positive pressures at the impact site, negative pressure at the site opposite of impact. Recently, Hardy et al. demonstrated the presences of transient pressure pulses with impact conditions. Coup pressures measured within a pressurized cadaver head after impact ranged from 34 to 160 kPa, and the contrecoup pressures ranged from −2 to −48 kPa [3]. Pamela et al. tested the effect of overpressure from positive pressure to negative pressure on astrocytes. Pressure wave generated by the barochamber, with high amplitude and short duration in the first pulse [4]. However, there is a lack of information with regards to the effect of impact pressure on endothelial cells in vitro, which are the components of BBB.© 2012 ASME

Strain Magnitude and Strain Rate Influence Stretch-Induced Injury of PC12 Cells

The acceleration-deceleration associated with external impact to head causes the mechanical deformation of brain tissue, resulting in neuronal damage. However, the correlation between the deformation and the neuronal damage has not been understood sufficiently. In this study, we investigated the effects of various magnitudes and rates of strain on cytotoxicity, mortality and morphology of Rat pheochromocytoma cell line, PC12 cells using the shock machine. PC12 cells were seeded in poly-L-lysine-coated polycarbonate dishes and neurites were elongated by the addition of neuron growth factor. The dishes were weighted at the bottom of 0.2 mm thickness in order to strain it by loading acceleration induced by the shock machine. The magnitude of acceleration was 500 G and the duration of acceleration was 1 ms. Then, PC12 cells were subjected to strain magnitudes (up to 2.2%) and strain rates (up to 19.0 (-1)). After exposure to impact, lactate dehydrogenase (LDH) was measured as cytotoxicity and cell viability was measured by the dye exclusion method with trypan blue dye as cell mortality. Morphology of neurites in PC12 cells were observed by phase contrast microscopy. As a result, cytotoxicity and cell mortality increased with increase in strain magnitude and strain rate. In addition, swelling and beading of neurites in PC12 cells increased with increase in strain magnitudes or strain rates. These results suggested that stretch-induced injury of PC12 cells is dependent on strain magnitude and strain rate, but, in order to elucidate neuronal damage caused by an interaction between strain magnitude and strain rate, more quantitative studies are required, such as various combination of strain magnitude and strain rate.

Finite element head model simulation of the case suspected of diffuse axonal injury in the traffic accident

ABSTRACT A traumatic brain injury case that diffuses axonal injury was suspected in the physicians diagnosis was simulated using finite element head model based on medical information such as MRI and CT. First, the head model matched the subjects head dimensions and material properties of the skull from the subjects age. Next, the site of the subjects head collision was estimated from the contusion, and collision velocity was estimated from the presence or absence of skull fractures and from the presence or absence of intracranial microhaemorrhage. Finally, we predicted the region of onset and severity of the neurological injury from intracerebral von Mises stress, and the analytical result showed the possibility of mild and partly moderate neurological injury. Furthermore, we assessed the effect of each parameter on the severity of the neurological injury and ascertained that collision velocity is the factor with the greatest effect on von Mises stress.

Prediction of Traumatic Brain Injuries by FEM Through Accident Reconstructions

In the head injury accidents, the injuries such as a bone fracture, bleeding or the cerebral contusion can easily be observed by the primary care and quick treatment is possible, but the nerve axonal damage is hard to be discovered and treatment tends to be late. In this paper, the method that predicts the detailed traumatic brain injury including the axonal injury by computer simulation based on emergency medical treatment data are presented and the items which are indispensable for the computer simulation in the medical data are shown. The medical data of 213 head injury accidents were provided by the hospital and they were classified into two groups, the group which can be reconstructed and the group which cannot be reconstructed. It was judged that 10 cases of the traffic accident of all 134 cases can be reconstructed and 23 cases of the fall/fall down accidents of all 61 cases can be reconstructed. As examples, the typical traffic accident and the fall down accident cases were introduced, and the reconstruction process of the accidents and the result of the brain injury prediction are explained in detail.

Repetitive stretching enhances the formation of neurite swellings in cultured neuronal cells

Repetitive mild traumatic brain injury (r-mTBI) has been gaining increasing attention from the researchers since several studies have reported that the cognitive dysfunctions after single mTBI become measurably long-term deficits, such as delayed speed of processing and memory dysfunction, after r-mTBI, contributing to the emerging hypothesis that r-mTBI may cause cumulative damage to the brain, and in the absence of cell death, could result in cognitive deficits which may ultimately progress to memory and learning dysfunction. Studies also associated r-mTBI with “second-impact syndrome” and chronic traumatic encephalopathy (CTE) as possible consequences of r-mTBI. However, the potential injury mechanisms involved in r-mTBI still remain unclear and research on rmTBI is still in early stages. Thereby, in this study an in vitro uniaxial stretching model was used to investigate the r-mTBI related cell damage for clarifying the pathology and post-injury sequelae of rm TBI in comparison with single mTBI. 3 types of stretching experiments were conducted including the single loading groups that were subjected once to stretching with a strain of 0.1 at a strain rate of 5 s-1, the repetitive loading groups that were subjected again to the same stretching 1 h after the initial stretching, and the sham control groups that were set in and removed from the uniaxial stretching device without receiving mechanical loading. Results shows that even though initial insult induced some level of swelling formation, swelling formation increased by second stretching confirming that r-mTBI causes increased amounts of cellular damage when compared with single insults of the same magnitude. Moreover, the absence of progression to cell death at 24 h post injury is detected after swelling formation by repetitive stretching. These data indicate that if injured neurons are subsequently subjected to low strain at the same level, the rate of injury significantly increases confirming the emerging hypothesis on repetitive mTBI exacerbating traumatic axonal injury. Background To this day, traumatic brain injury (TBI) continues to be a leading cause of mortality and morbidity worldwide, making TBI a significant public health problem [1]. In the United States alone, the estimated number for annual occurrence of TBI is 1,700,000 [2-3] whereas in Asia 300-400, and in European countries (average of the 23 countries) 200-300 people per 100 000 are hospitalized annually due to TBI [46]. Moreover, majority of these cases categorized as mild TBI (mTBI) or concussion [2,7] while, in spite of the name “mild”, approximately 15% of mTBI patients suffering persistent cognitive dysfunction in the United States alone [8]. Furthermore, several studies on professional athletes and military personnel have reported that these cognitive dysfunctions become measurably long-term deficits, such as delayed speed of processing and memory dysfunction, after “repetitive” mTBI (r-mTBI), contributing to the emerging hypothesis that r-mTBI may cause cumulative damage to the brain, and in the absence of cell death, could result in cognitive deficits which may ultimately progress to memory and learning dysfunction [9-17]. Aiding to the human cases, studies on animal models have also demonstrated a worsened outcome with repetitive TBI, where cell death and tissue damage are common consequences of moderate and severe TBI, not mTBI, yet cellular dysfunction and memory deficits are overt after mTBI [18-22]. Additionally, increasing evidence suggests that mTBI patients suffer from diffuse axonal injury (DAI), which is one of the most common pathology of TBI, associated with rapid brain deformation, stretching, inertial forces occurring as a result of traumatic incidents such as accidents, falls and assaults resulting in the stretching of neuronal axons [23-27] where primary damage to axons progressively develops into secondary cascades such as neuronal degeneration and axonal cytoskeletal disconnection [28-31]. Considering that DAI is involved in the immediate loss of conscioussness after TBI and several studies have been shown white matter abnormalities consistent with DAI in mTBI patients [32-34], there is a strong possibility of a potential mechanism which leads to vulnerability with a repeat injury since phenotypic or physiologic changes of the injured axons would likely influence outcome following r-mTBI and that DAI has an important role in r-mTBI. Studies also associated r-mTBI with “secondimpact syndrome” where individuals displaying post-concussion symptoms after initial TBI, which can include visual, sensory or motor dysfunctions or mental difficulty such as cognitive and memory problems, and devastating brain swelling resulting in semicomatose situation with dilating pupils, loss of eye movement and respiratory failure after a second TBI occurring days or weeks after the first injury [35]. More recently, similar pathology, referred to as chronic traumatic encephalopathy (CTE), a neurodegenerative brain disorder, has been gaining growing awareness as another consequence of r-mTBI [36-40]. However, the potential injury mechanisms involved in r-mTBI still remain unclear and research on r-mTBI is still in early stages. Although in vivo models contribute substantially in understanding pathological and physiological sequelae on macroscopic and microscopic levels, complementing these with in vitro studies that simulate specific Correspondence to: Hiromichi Nakadate, Graduate School of System Design, Tokyo Metropolitan University, Tokyo, Japan, E-mail: nakadate@tmu.ac.jp

An in Vitro Stretch-Injury Model for Elongation-Controlled Neuronal Cells: Effect of Strains Along Neurite

Diffuse axonal injury (DAI), a major component of traumatic brain injury, is associated with rapid deformation of brain tissue resulting in the stretching of neural axons. Focal axonal beading, which is the morphological hallmarks of DAI pathology, leads to the disconnection of neurons from tissues, resulting in cell death. Our goal is better understanding of neuronal tolerance and help to predicting the pathogenesis of DAI from mechanical loading to the head. In present study, we developed a stretch-injury model that subjected cultured neuronal cells in which the directions of neurite elongation were controlled with microfluidic culture technique to uniaxial stretch and examined the effect of strains along neurite on the cell damage. Neurites from PC 12 cells were extended at 0, 45 and 90 degrees to stretch direction using a fabricated poly(dimethylsiloxane) (PDMS) piece having microgrooves in combination with PDMS substrate. Following stretch with a strain of 0.22 and a strain rate of 27 s− 1, the morphology of same neurites were observed at 5 min-24 h. As a result, the beading along neurites oriented at 0 degree increased immediately following stretch and the increase was sustained until 24 h, although the beading along neurites oriented at 45 and 90 degrees transiently increased within 1 h following stretch. Many rupture of neurites was observed more in neurites oriented at 0 and 90 degrees than in neurites oriented at 45 degrees, however, the retraction and disappearance of neurites did not differ in any orientation conditions. These results suggest that the difference in strain along neurite induces different types and degrees of neurite damage and not strain of cellcultured substrate but strain loaded to neurite is important to evaluation of neuronal injury.

Study on the Mechanism of Traumatic Brain Injury

Skull fracture, intracranial hemorrhage, or cerebral injury can be caused in humans due to a strong impact to the head. The following 2 types of cerebral injuries are often observed: one type is cerebral contusion which is a local brain damage to the brain, and the other is diffuse axonal injury (DAI) which is a diffuse brain damage to the brain. In various head injuries caused by external impact, cerebral contusion and DAI mainly result in direct failure of the cerebral parenchyma.