Mattias K. Sköld

Induction of VEGF and VEGF receptors in the spinal cord after mechanical spinal injury and prostaglandin administration.

Vascular endothelial growth factor (VEGF) is an angiogenetic factor that promotes endothelial cell proliferation during development and after injury to various types of tissue, including the central nervous system (CNS). Using immunohistochemical and in situ hybridization methods we have here demonstrated that VEGF and its receptors Flk‐1, Flt‐1 and Neuropilin‐1 mRNAs and proteins are induced after incisions in the rat spinal cord. The inducible enzyme for prostaglandin synthesis cyclooxygenase‐2 (COX‐2) is known to be upregulated after spinal injury, cerebral ischemia and to stimulate angiogenesis. To test the hypothesis that prostaglandins may be involved in the VEGF response after lesion we investigated whether intraspinal microinjections of prostaglandin F2α (PGF2α) alters VEGF expression in the spinal cord. Such treatment was followed by a strong upregulation of VEGF mRNA and protein in the injection area. Finally, by use of an in vitro model with cell cultures of meningeal fibroblast and astrocyte origin, resembling the lesion area cellular content after spinal cord injury but devoid of inflammatory cells, we showed that VEGF is expressed in this in vitro model cell system after treatment with PGF2α and prostaglandin E2 (PGE2). These data suggest that cells within a lesion area in the spinal cord are capable of expressing VEGF and its receptors in response to mechanical injury and that prostaglandins may induce VEGF expression in such cells, even in the absence of inflammatory cells.

Inhibition of vascular endothelial growth factor receptor 2 activity in experimental brain contusions aggravates injury outcome and leads to early increased neuronal and glial degeneration

Angiogenesis following traumatic brain injuries (TBIs) may be of importance for post‐traumatic reparative processes and the development of secondary injuries. We have previously shown expression of vascular endothelial growth factor (VEGF), a major regulator of endothelial cell proliferation, angiogenesis and vascular permeability, and VEGF receptors (VEGFR1 and 2) after TBI in rat. In the present work we tried to further elucidate the role of VEGF after TBI by performing specific VEGFR2 activity inhibition. In rats subjected to VEGFR2 blockage we report an increased haemorrhagic area (P < 0.05), early increase in serum levels of neural injury marker neuron‐specific enolase (P < 0.05) and glial injury marker S100β (P < 0.05), and increased numbers of terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate‐biotin nick end labelling‐ (TUNEL‐) and FluoroJade B‐ (P < 0.05) positive cells, all increases preceding the known VEGF/VEGFR vascular response in brain trauma. An increase in lesion area, as measured by decreased microtubuli‐associated protein 2 expression (P < 0.05) and increased glial fibrillary acidic protein reactivity (P < 0.05), could also be demonstrated. In addition, vascular density, as measured by von Willebrandt factor‐positive cells, was decreased (P < 0.05). No differences in post‐traumatic inflammatory response, as measured by stainings for macrophages, granulocytes and intracellular adhesion molecules, were shown between the groups. Taken together, our findings point towards VEGF/VEGFR2 up‐regulation after TBI as being an important endogenous cytoprotective mechanism in TBI. The possible importance of VEGF on the vascular, neuronal and glial compartments of the neurovascular unit after TBI is discussed.

Activating Transcription Factor 3, a Useful Marker for Regenerative Response after Nerve Root Injury

Activating transcription factor 3 (ATF3) is induced in various tissues in response to stress. In this experiment, ATF3 expression was studied in adult rats subjected either to a dorsal or ventral root avulsion (VRA; L4-6), or sciatic nerve transection (SNT). Post-operative survival times varied between 1.5 h and 3 weeks. In additional experiments an avulsed ventral root was directly replanted to the spinal cord. Dorsal root ganglias (DRGs) from humans exposed to traumatic dorsal root avulsions were also examined. After SNT ATF3 immunoreactivity (ATF3 IR) was detected in a few DRG neurons already 6 h after the lesion. After 24 h the number had clearly increased and still at 3 weeks DRG neurons remained labeled. In the ventral horn, ATF3 IR in motoneurons (MN) was first detected 24 h after the SNT, and still 3 weeks post-operatively lesioned MN showed ATF3 labeling. After a VRA many spinal MN showed ATF3 IR already after 3 h, and after 6 h all MN were labeled. At 3 weeks a majority of the lesioned MN had died, but all the remaining ones were labeled. When an avulsed ventral root was directly replanted, MN survived and were still labeled at 5 weeks. In DRG, a few neurons were labeled already at 1.5 h after a dorsal root avulsion. At 24 h the number had increased but still only a minority of the neurons were labeled. At 3 days the number of labeled neurons was reduced, and a further reduction was at hand at 7 days and 3 weeks. In parallel, in humans, 3 days after a traumatic dorsal root avulsion, only a few DRG neurons showed ATF3 IR. At 6 weeks no labeled neurons could be detected. These facts imply that ATF3 response to axotomy involves a distance-dependent mechanism. ATF3 also appears to be a useful and reliable neuronal marker of nerve lesions even in humans. In addition, ATF3 up-regulation in both motor and sensory neurons seems to be linked to regenerative competence.

Induction of HIF1α but not HIF2α in motoneurons after ventral funiculus axotomy—implication in neuronal survival strategies

Spinal cord injury is frequently associated with local tissue hypoxia. As neuronal cells are susceptible to damage caused by low oxygen levels, hypoxia-induced activation of tissue-protective factors could represent an endogenous mechanism for neuron survival following injury. We studied in vivo, in a rat model of intraspinal axotomy of motoneurons, the cell- and time-dependent regulation of the hypoxia-inducible transcription factors (HIFs), HIF1alpha and HIF2alpha, as well as one of their target genes, vascular endothelial growth factor (VEGF). VEGF is a potent hypoxia-regulated angiogenic growth factor with recently discovered neuroprotective and neurotrophic activities. While neither HIF1alpha, HIF2alpha, nor VEGF mRNA were detected in noninjured motoneurons, we found a strong induction of HIF1alpha, but not HIF2alpha mRNA in axotomized motoneurons. HIF1alpha expression peaked at about 7 days after injury. Moreover, we found increased VEGF mRNA and protein expression around and within the scar but also within motoneurons, peaking around 3 days after axotomy. In addition, increased survival of cultured motoneurons after treatment with VEGF could also be shown. We conclude that axotomized motoneurons in this model respond to injury by specific induction of HIF1alpha and VEGF expression that may provide an endogenous mechanism with the potential to promote motoneuron survival after injury.

On Acute Gene Expression Changes after Ventral Root Replantation

Replantation of avulsed spinal ventral roots has been show to enable significant and useful regrowth of motor axons in both experimental animals and in human clinical cases, making up an interesting exception to the rule of unsuccessful neuronal regeneration in central nervous system. Compared to avulsion without repair, ventral root replantation seems to rescue lesioned motoneurons from death. In this study we have analyzed the acute response to ventral root avulsion and replantation in adult rats with gene arrays combined with cluster analysis of gene ontology search terms. The data show significant differences between rats subjected to ventral replantation compared to avulsion only. Even though number of genes related to cell death is similar in the two models after 24 h, we observed a significantly larger number of genes related to neurite growth and development in the rats treated with ventral root replantation, possibly reflecting the neuroregenerative capacity in the replantation model. In addition, an acute inflammatory response was observed after avulsion, while effects on genes related to synaptic transmission were much more pronounced after replantation than after avulsion alone. These data indicate that the axonal regenerative response from replantation is initiated at an earlier stage than the possible differences in terms of neuron survival. We conclude that this type of analysis may facilitate the comparison of the acute response in two types of 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.

Karolinska Institutet 200-Year Anniversary. Symposium on Traumatic Injuries in the Nervous System: Injuries to the Spinal Cord and Peripheral Nervous System – Injuries and Repair, Pain Problems, Lesions to Brachial Plexus

The Karolinska Institutet 200-year anniversary symposium on injuries to the spinal cord and peripheral nervous system gathered expertise in the spinal cord, spinal nerve, and peripheral nerve injury field spanning from molecular prerequisites for nerve regeneration to clinical methods in nerve repair and rehabilitation. The topics presented at the meeting covered findings on adult neural stem cells that when transplanted to the hypoglossal nucleus in the rat could integrate with its host and promote neuron survival. Studies on vascularization after intraspinal replantation of ventral nerve roots and microarray studies in ventral root replantation as a tool for mapping of biological patterns typical for neuronal regeneration were discussed. Different immune molecules in neurons and glia and their very specific roles in synapse plasticity after injury were presented. Novel strategies in repair of injured peripheral nerves with ethyl-cyanoacrylate adhesive showed functional recovery comparable to that of conventional epineural sutures. Various aspects on surgical techniques which are available to improve function of the limb, once the nerve regeneration after brachial plexus lesions and repair has reached its limit were presented. Moreover, neurogenic pain after amputation and its treatment with mirror therapy were shown to be followed by dramatic decrease in phantom limb pain. Finally clinical experiences on surgical techniques to repair avulsed spinal nerve root and the motoric as well as sensoric regain of function were presented.

Expression of Semaphorins, Neuropilins, VEGF, and Tenascins in Rat and Human Primary Sensory Neurons after a Dorsal Root Injury

Dorsal root injury is a situation not expected to be followed by a strong regenerative growth, or growth of the injured axon into the central nervous system of the spinal cord, if the central axon of the dorsal root is injured but of strong regeneration if subjected to injury to the peripherally projecting axons. The clinical consequence of axonal injury is loss of sensation and may also lead to neuropathic pain. In this study, we have used in situ hybridization to examine the distribution of mRNAs for the neural guidance molecules semaphorin 3A (SEMA3A), semaphorin 3F (SEMA3F), and semaphorin 4F (SEMA4F), their receptors neuropilin 1 (NP1) and neuropilin 2 (NP2) but also for the neuropilin ligand vascular endothelial growth factor (VEGF) and Tenascin J1, an extracellular matrix molecule involved in axonal guidance, in rat dorsal root ganglia (DRG) after a unilateral dorsal rhizotomy (DRT) or sciatic nerve transcetion (SNT). The studied survival times were 1–365 days. The different forms of mRNAs were unevenly distributed between the different size classes of sensory nerve cells. The results show that mRNA for SEMA3A was diminished after trauma to the sensory nerve roots in rats. The SEMA3A receptor NP1, and SEMA3F receptor NP2, was significantly upregulated in the DRG neurons after DRT and SNT. SEMA4F was upregulated after a SNT. The expression of mRNA for VEGF in DRG neurons after DRT showed a significant upregulation that was high even a year after the injuries. These data suggest a role for the semaphorins, neuropilins, VEGF, and J1 in the reactions after dorsal root lesions.

Editorial: When Physics Meets Biology; Biomechanics and Biology of Traumatic Brain Injury

The Editorial on the Research Topic When Physics Meets Biology; Biomechanics and Biology of Traumatic Brain Injury We have launched this special topic to focus on the critical relationships between physical forces, biomechanics, and biological responses in traumatic brain injury (TBI). Understanding the precise connection between physical force(s) and biological response(s), the “physical to biological coupling” is essential for developing high fidelity models in experimental TBI, for designing better protective devices and measures, for establishing more accurate diagnostics, and – most importantly – for identifying specific, evidence-based pharmacotherapies for TBI. Several of the contributions are dedicated to the very important topic of modeling various forms of brain trauma describing an in vitro model of cavitation (Cao et al.), models of mild TBI (Chen et al.). Blast-induced TBI poses special challenge for modeling and two reviews are dedicated to address some of the issues associated with blast-induced TBI research (Courtney and Courtney; Needham et al.). As US epidemiology data show, the importance of understanding penetrating TBI – sadly – has became even more urgent. Cernak et al. describe a novel model for penetrating TBI, whereas Davidsson and Risling provide a great example of using finite element modeling in penetrating TBI. This study, together with a review by Carlsen and Daphalapurkar, focusing on the importance of structural anisotropy in biofidelity of computational models, both include the possibility for more sophisticated material definitions and can implement increasingly more physiologically relevant measures of injury. The overview by Young et al. fills a critical gap by comparing the physics of high velocity penetrating, blunt impact and blast injuries. As these contributions illustrate, modeling TBI is a truly multidimensional problem. The first dimension, the physical forces are highly variable and complex. They vary from relatively low-speed, low kinetic energy, typical in traffic and sport accidents to high velocity, high kinetic energy type observed in explosive blast. The next dimension is how these forces interact with the head and ultimately with the brain, whether they interact with the entire head during acceleration–deceleration causing diffuse, closed head TBI, or if they are high velocity, concentrated kinetic energy propelling, e.g., a bullet penetrating the skull and brain parenchyma causing focal, open TBI, or the combination of various velocities and intensities. The next important, yet currently, understudied dimension in modeling is to take into account the directionality of the physical forces relative to the anatomy of the brain. The use of the new generation of sensors, such as the Vector mouth guard (i1 Biometrics, Inc., Kirkland, WA, USA) or the Prevent™ Concussion Impact Monitor Mouthguard (Prevent Biometrics, MN, USA) in combination with functional and molecular outcome measures can provide data not only about the g-forces but also about vectorial information in human/clinical TBI. Precise readout of g-forces and their directionality relative to neuroanatomical structures coupled with neurobehavioral and molecular outcome measures has the potential to elevate current sensor technology to a whole new level. While the biological responses to the various physical forces are quite similar, there are pathological changes of that are predominantly triggered by specific physical force. Such example is axonal injury in response to rotational forces. Most pathobiologies of the secondary injury process, metabolic changes, vascular damage, inflammation, etc., seem to occur independent of the physical force or directionality. However, the sequelae, the onset, and extent of the individual pathologies appear to be affected by the causative physical force. It is, thus, conceivable that future sensors using “big data” approaches will be able to provide predictive information about the biological response to the physical impact. Such system will be able to identify individuals with increased cerebral vulnerability and will guide safe return to play or duty. Similarly, such advanced sensor system will be able to recommend personalized treatments taking into account the injured individual’s biology, medical history, etc. Unfortunately, no similar sensors are currently in use in experimental TBI. This further increases the gap between experimental and clinical TBI studies; in clinical TBI, we can determine the extent of functional deficits and using the new generation of sensors, we can increasingly measure the causative physical forces. In experimental TBI, on the other hand, we set the indirect parameters of the injury (e.g., pressure in fluid percussion) but we do not measure the head movement (where applicable) and only infrequently the type and extent of the functional deficits caused by the indirect physical forces. Our hope is that the new knowledge derived from the better understanding of “physical to biological coupling” will also lead to improved helmet design, car safety, etc. We also hope that the ability to connect the physics to biological outcomes will help to classify TBI cases based on the physical forces, directionality, and biological responses, rather than the currently used Glasgow Coma scale (GCS). The failure to develop specific and effective pharmacotherapies in TBI, despite of the many promising preclinical studies, may partly stem from lack of understanding and modeling of the very physical forces that trigger the specific secondary process. We hope that this research topic will contribute toward closing the gap between experimental and clinical TBI studies. We believe that such a “back to basics” approach is much needed and warranted and will help researchers, and the many new scientists entering the field, to better understand the physics and the biology of TBI so to better select appropriate models to their research. We believe that the current models of TBI need refinement and that the first step toward high fidelity modeling is the understanding of the physics and the physical forces. We also believe that only those models should be used that mirrors the physical forces causing human TBI. Lastly, but most importantly, we hope that our effort will contribute to provide better care to victims of TBI in the form of specific diagnosis and treatments. In the age of “wearable devices” that can monitor a multitude of parameters connected by of “Internet of things,” and analyzed by using “big data” approaches, it is not too far fetched to see a completely novel ways coming “online” to model, diagnose, and treat TBI.

Neurite Growth and Polarization on Vitronectin Substrate after in Vitro Trauma is not Enhanced after IGF Treatment

Following traumatic brain injuries (TBI), insulin-like growth factor (IGF) is cortically widely upregulated. This upregulation has a potential role in the recovery of neuronal tissue, plasticity, and neurotrophic activity, though the molecular mechanisms involved in IGF regulation and the exact role of IGF after TBI remain unclear. Vitronectin (VN), an extracellular matrix (ECM) molecule, has recently been shown to be of importance for IGF-mediated cellular growth and migration. Since VN is downregulated after TBI, we hypothesized that insufficient VN levels after TBI impairs the potential beneficial activity of IGF. To test if vitronectin and IGF-1/IGFBP-2 could contribute to neurite growth, we cultured hippocampal neurons on ± vitronectin-coated coverslips and them treated with ± IGF-1/IGF binding protein 2 (IGFBP-2). Under same conditions, cell cultures were also subjected to in vitro trauma to investigate differences in the posttraumatic regenerative capacity with ± vitronectin-coated coverslips and with ± IGF-1/IGFBP-2 treatment. In both the control and trauma situations, hippocampal neurons showed a stronger growth pattern on vitronectin than on the control substrate. Surprisingly, the addition of IGF-1/IGFBP-2 showed a decrease in neurite growth. Since neurite growth was measured as the number of neurites per area, we hypothesized that IGF-1/IGFBP-2 contributes to the polarization of neurons and thus induced a less dense neurite network after IGF-1/IGFBP-2 treatment. This hypothesis could not be confirmed and we therefore conclude that vitronectin has a positive effect on neurite growth in vitro both under normal conditions and after trauma, but that addition of IGF-1/IGFBP-2 does not have a positive additive effect.