Stefan Plantman

Mechanisms of blast induced brain injuries, experimental studies in rats

Traumatic brain injuries (TBI) potentially induced by blast waves from detonations result in significant diagnostic problems. It may be assumed that several mechanisms contribute to the injury. This study is an attempt to characterize the presumed components of the blast induced TBI. Our experimental models include a blast tube in which an anesthetized rat can be exposed to controlled detonations of explosives that result in a pressure wave with a magnitude between 130 and 260 kPa. In this model, the animal is fixed with a metal net to avoid head acceleration forces. The second model is a controlled penetration of a 2mm thick needle. In the third model the animal is subjected to a high-speed sagittal rotation angular acceleration. Immunohistochemical labeling for amyloid precursor protein revealed signs of diffuse axonal injury (DAI) in the penetration and rotation models. Signs of punctuate inflammation were observed after focal and rotation injury. Exposure in the blast tube did not induce DAI or detectable cell death, but functional changes. Affymetrix Gene arrays showed changes in the expression in a large number of gene families including cell death, inflammation and neurotransmitters in the hippocampus after both acceleration and penetration injuries. Exposure to the primary blast wave induced limited shifts in gene expression in the hippocampus. The most interesting findings were a downregulation of genes involved in neurogenesis and synaptic transmission. These experiments indicate that rotational acceleration may be a critical factor for DAI and other acute changes after blast TBI. The further exploration of the mechanisms of blast TBI will have to include a search for long-term effects.

Impeded Interaction between Schwann Cells and Axons in the Absence of Laminin α4

The Schwann cell basal lamina (BL) is required for normal myelination. Loss or mutations of BL constituents, such as laminin-2 (α2β1γ1), lead to severe neuropathic diseases affecting peripheral nerves. The function of the second known laminin present in Schwann cell BL, laminin-8 (α4β1γ1), is so far unknown. Here we show that absence of the laminin α4 chain, which distinguishes laminin-8 from laminin-2, leads to a disturbance in radial sorting, impaired myelination, and signs of ataxia and proprioceptive disturbances, whereas the axonal regenerative capacity is not influenced. In vitro studies show poor axon growth of spinal motoneurons on laminin-8, whereas it is extensive on laminin-2. Schwann cells, however, extend longer processes on laminin-8 than on laminin-2, and, in contrast to the interaction with laminin-2, solely use the integrin receptor α6β1 in their interaction with laminin-8. Thus, laminin-2 and laminin-8 have different critical functions in peripheral nerves, mediated by different integrin receptors.

Integrin-laminin interactions controlling neurite outgrowth from adult DRG neurons in vitro

A prerequisite for axon regeneration is the interaction between the growth cone and the extracellular matrix (ECM). Laminins are prominent constituents of ECM throughout the body, known to support axon growth in vitro and in vivo. The regenerative capacity of adult neurons is greatly diminished compared to embryonic or early postnatal neurons. Since most lesions in the nervous system occur in the adult, we have examined neurite outgrowth from adult mouse DRG neurons on four laminin isoforms (laminin-1/LM-111, laminin-2/LM-211, laminin-8/LM-411 and laminin-10/LM-511) in vitro. The growth on laminin-1 and -10 was trophic factor-independent and superior to the one on laminin-2 and -8, where growth was very poor in the absence of neurotrophins. Among other ECM proteins, laminins were by far the most active molecules. Using function-blocking antibodies to laminin-binding integrins, we identified non-overlapping functions of integrins alpha3beta1, alpha7beta1 and alpha6beta1 on different laminin isoforms, in that alpha3beta1 and alpha7beta1 integrins appeared to be specific receptors for both laminin-1 and-2, whereas integrin alpha6beta1 was a receptor for laminin-8 and-10. Lastly, by use of immunohistochemistry, expression of subunits of laminin-1, -2, -8 and -10 in sensory organs in the human epidermis could be demonstrated, supporting an important role for these laminins in relation to primary sensory axons.

A Model for Mild Traumatic Brain Injury that Induces Limited Transient Memory Impairment and Increased Levels of Axon Related Serum Biomarkers

Mild traumatic brain injury (mTBI) is one of the most common neuronal insults and can lead to long-term disabilities. mTBI occurs when the head is exposed to a rapid acceleration-deceleration movement triggering axonal injuries. Our limited understanding of the underlying pathological changes makes it difficult to predict the outcome of mTBI. In this study we used a scalable rat model for rotational acceleration TBI, previously characterized for the threshold of axonal pathology. We have analyzed whether a TBI just above the defined threshold would induce any detectable behavioral changes and/or changes in serum biomarkers. The effect of injury on sensory motor functions, memory and anxiety were assessed by beam walking, radial arms maze and elevated plus maze at 3–7 days following TBI. The only behavioral deficits found were transient impairments in working and reference memory. Blood serum was analyzed at 1, 3, and 14 days after injury for changes in selected protein biomarkers. Serum levels of neurofilament heavy chain and Tau, as well as S100B and myelin basic protein showed significant increases in the injured animals at all time points. No signs of macroscopic injuries such as intracerebral hematomas or contusions were found. Amyloid precursor protein immunostaining indicated axonal injuries at all time points analyzed. In summary, this model mimics some of the key symptoms of mTBI, such as transient memory impairment, which is paralleled by an increase in serum biomarkers. Our findings suggest that serum biomarkers may be used to detect mTBI. The model provides a suitable foundation for further investigation of the underlying pathology of mTBI.

Dorsal root ganglion neurons up-regulate the expression of laminin-associated integrins after peripheral but not central axotomy.

The favorable prognosis of regeneration in the peripheral nervous system after axonal lesions is generally regarded as dependent on the Schwann cell basal lamina. Laminins, a heterotrimeric group of basal lamina molecules, have been suggested to be among the factors playing this supportive role. For neurons to utilize laminin as a substrate for growth, an expression of laminin binding receptors, integrins, is necessary. In this study, we have examined the expression of laminin binding integrin subunits in dorsal root ganglion (DRG) neurons after transection to either their peripherally projecting axons, as in the sciatic nerve, followed by regeneration, or the centrally projecting axons in dorsal roots, followed by no or weak regenerative activity. In uninjured DRG, immunohistochemical staining revealed a few neurons expressing integrin subunit α6, whereas integrin subunits α7 and foremost β1 were expressed in a majority of neurons. After an injury to the sciatic nerve, mRNAs encoding all three integrins were up‐regulated in DRG neurons. By anterograde tracing, immunoreactivity for all studied integrins was also found in association with growing axons after a sciatic nerve crush lesion in vivo. In contrast, mRNA levels remained constant in DRG neurons after a dorsal root injury. Together with previous findings, this suggests that integrin subunits α6, α7, and β1 have an important role in the regenerative response following nerve injury and that the lack of regenerative capacity following dorsal root injury could in part be explained by the absence of response in integrin regulation. J. Comp. Neurol. 480:162–169, 2004.

Classical Major Histocompatibility Complex Class I Molecules in Motoneurons: New Actors at the Neuromuscular Junction

Major histocompatibility complex (MHC) class I molecules have fundamental functions in the immune system. Recent studies have suggested that these molecules may also have non-immune functions in the nervous system, in particular related to synaptic function and plasticity. Because adult motoneurons express mRNAs for MHC class I molecules, we have examined their subcellular expression pattern in vivo and their role for the synaptic connectivity of these neurons. We observed immunoreactivity for classical MHC class I (Ia) protein in motoneuron somata, but the predominant expression was found in axons and presynaptically at neuromuscular junctions (NMJs). Peripheral nerve lesion induced a strong increase of motoneuron MHC class Ia (H2-Kb/Db) mRNA, indicating a role for MHC class Ia molecules during regeneration. Accordingly, there was an accumulation of MHC class Ia proteins at the cut ends and in growth cones of motor axons after lesion. In Kb−/−Db−/− mice (lacking MHC class Ia molecules), the time course for recovery of grip ability in reinnervated muscles was significantly delayed. Muscles from Kb−/−Db−/− mice displayed an increased density and a disturbed distribution of NMJs and fewer terminal Schwann cells/NMJ compared with wild-type mice. A population of Schwann cells in sciatic nerves expressed the paired Ig receptor B, which binds to MHC class I molecules. These results provide the first evidence that neuronal MHC class Ia molecules are present in motor axons, that they are important for organization of NMJs and motor recovery after nerve lesion, and that their actions may be mediated via Schwann cells.

Characterization of a Novel Rat Model of Penetrating Traumatic Brain Injury

A penetrating traumatic brain injury (pTBI) occurs when an object impacts the head with sufficient force to penetrate the skin, skull, and meninges, and inflict injury directly to the brain parenchyma. This type of injury has been notoriously difficult to model in small laboratory animals such as rats or mice. To this end, we have established a novel non-fatal model for pTBI based on a modified air rifle that accelerates a pellet, which in turn impacts a small probe that then causes the injury to the experimental animals brain. In the present study, we have focused on the acute phase and characterized the tissue destruction, including increasing cavity formation, white matter degeneration, hemorrhage, edema, and gliosis. We also used a battery of behavioral models to examine the neurological outcome, with the most noteworthy finding being impairment of reference memory function. In conclusion, we have described a number of events taking place after pTBI in our model. We expect this model will prove useful in our efforts to unravel the biological events underlying injury and regeneration after pTBI and possibly serve as a useful animal model in the development of novel therapeutic and diagnostic approaches.

The Loop Diuretic Bumetanide Blocks Posttraumatic p75NTR Upregulation and Rescues Injured Neurons

Injured neurons become dependent on trophic factors for survival. However, application of trophic factors to the site of injury is technically extremely challenging. Novel approaches are needed to circumvent this problem. Here, we unravel the mechanism of the emergence of dependency of injured neurons on brain-derived neurotrophic factor (BDNF) for survival. Based on this mechanism, we propose the use of the diuretic bumetanide to prevent the requirement for BDNF and consequent neuronal death in the injured areas. Responses to the neurotransmitter GABA change from hyperpolarizing in intact neurons to depolarizing in injured neurons. We show in vivo in rats and ex vivo in mouse organotypic slice cultures that posttraumatic GABAA-mediated depolarization is a cause for the well known phenomenon of pathological upregulation of pan-neurotrophin receptor p75NTR. The increase in intracellular Ca2+ triggered by GABA-mediated depolarization activates ROCK (Rho kinase), which in turn leads to the upregulation of p75NTR. We further show that high levels of p75NTR and its interaction with sortilin and proNGF set the dependency on BDNF for survival. Thus, application of bumetanide prevents p75NTR upregulation and neuronal death in the injured areas with reduced levels of endogenous BDNF.

Alteration in BDNF and its receptors, full-length and truncated TrkB and p75NTR following penetrating traumatic brain injury

The evidence that BDNF is involved in neuroprotection, neuronal repair and recovery after traumatic brain injury (TBI) is substantial. We have previously shown that the polymorphism of the human BDNF gene predicts cognitive recovery and outcome following penetrating TBI. The distribution of expression of BDNF and its receptors after penetrating TBI has not been investigated. In this study we examined the expression of these genes in a rat model of penetrating TBI. The injury is produced by a controlled penetration of a 2mm thick needle-shaped object, which is accelerated with a pellet from an air gun. We used in situ hybridization and investigated the mRNA expression of BDNF and its receptors: the full-length and the truncated TrkB and p75(NTR), from 1 day to 8 weeks following penetrating TBI. In addition, the protein level of BDNF in frontal cortex and hippocampus was measured by reverse phase protein microarray (RPPM). The mRNA expression of BDNF and its receptors decreased in the hippocampus in the border zone ipsilateral to the injury while there was an increase in mRNA expression at the contralateral side. The increase in BDNF mRNA expression in the hippocampus was sustained for 2 weeks following injury, with the highest expression noted in the CA3 cell layer. Furthermore, the protein analysis by RPPM showed increased levels of BDNF in the frontal cortex and the hippocampus up to 2 weeks after TBI. At 8 weeks following injury there was an intense labeling of the truncated TrkB receptor and the p75(NTR) in the area surrounding the cavity. Our study is the first report on the expression of BDNF and its receptors following penetrating TBI and suggests that their expression is altered long after the acute phase of injury. Further studies are needed to investigate if the late expressions of these receptors are beneficial or deleterious. In either case it indicates the possibility to influence the recovery after brain injury during the chronic phase and the development of treatments that may improve the outcome of TBI patients.

The Temporal Pattern of Changes in Serum Biomarker Levels Reveals Complex and Dynamically Changing Pathologies after Exposure to a Single Low-Intensity Blast in Mice.

Time-dependent changes in blood-based protein biomarkers can help identify the pathological processes in blast-induced traumatic brain injury (bTBI), assess injury severity, and monitor disease progression. We obtained blood from control and injured mice (exposed to a single, low-intensity blast) at 2-h, 1-day, 1–week, and 1-month post-injury. We then determined the serum levels of biomarkers related to metabolism (4-HNE, HIF-1α, ceruloplasmin), vascular function (AQP1, AQP4, VEGF, vWF, Flk-1), inflammation (OPN, CINC1, fibrinogen, MIP-1a, OX-44, p38, MMP-8, MCP-1 CCR5, CRP, galectin-1), cell adhesion and the extracellular matrix (integrin α6, TIMP1, TIMP4, Ncad, connexin-43), and axonal (NF-H, Tau), neuronal (NSE, CK-BB) and glial damage (GFAP, S100β, MBP) at various post-injury time points. Our findings indicate that the exposure to a single, low-intensity blast results in metabolic and vascular changes, altered cell adhesion, and axonal and neuronal injury in the mouse model of bTBI. Interestingly, serum levels of several inflammatory and astroglial markers were either unchanged or elevated only during the acute and subacute phases of injury. Conversely, serum levels of the majority of biomarkers related to metabolic and vascular functions, cell adhesion, as well as neuronal and axonal damage remained elevated at the termination of the experiment (1 month), indicating long-term systemic and cerebral alterations due to blast. Our findings show that the exposure to a single, low-intensity blast induces complex pathological processes with distinct temporal profiles. Hence, monitoring serum biomarker levels at various post-injury time points may provide enhanced diagnostics in blast-related neurological and multi-system deficits.