Interplay between blood-brain barrier disruption and neuroinflammation following severe traumatic brain injury

A severe traumatic brain injury (TBI) holds deleterious consequences for the afflicted, its next-of-kin and society. Still today, prognosis is semi-desolate. One explanation for this might be pathophysiological processes ensuing the primary trauma that are but indirectly targeted for treatment. Among such processes, blood-brain barrier (BBB) disruption and neuroinflammation constitute two astrocyte-dependent mechanisms that interplay in the aftermath of a severe TBI. The overall aim of this thesis was to characterize both BBB disruption and neuroinflammation translationally. In paper I, n = 17 patients with severe TBI were included in a prospective observational longitudinal study. Here, the protein biomarkers S100B and neuron-specific enolase (NSE) were sampled with high temporal resolution from both cerebrospinal fluid (CSF) and blood. We found that BBB disruption occurred among numerous patients and remained throughout the first week following injury. Interestingly, BBB disruption also affected clearance from brain to blood of S100B, but not NSE. This indicates that biomarkers are cleared differently from the injured CNS. We elaborated on this by utilizing a larger cohort size (n = 190 patients), which enabled outcome prediction modelling, in paper II. In this prospective, observational, cross-sectional study, we found that BBB disruption comprised a novel, independent outcome predictor that strongly related to levels of neuroinflammatory proteins in CSF and inflammatory processes within the injured brain. Among pathways assessed, particularly the complement system entailed proteins of future interest. We next assessed the relationship between in situ neuroinflammatory protein expression, BBB disruption, and brain edema in paper III. By utilizing a rodent model of severe TBI, we found that the cytotoxic edema region was associated with an innate neuroinflammatory response, and astrocytic aquaporin-4 retraction from the BBB interface. In fact, the astrocyte itself is an important neuroinflammatory cell, which we showed in paper IV, where we constructed a disease-modelling system of stem cell-derived astrocytes that we exposed to neuroinflammatory substances. Following neuroinflammatory stimulus, astrocytes exhibited an important increase in canonical stress-response pathways. Importantly, following stimulation with clinically relevant neuroinflammatory substances seen in human TBI from paper II, they also acquired a neurotoxic potential, of plausible importance for local cell survival following a severe TBI. Taken together, BBB disruption and neuroinflammation ensue a severe TBI. Neuroinflammation, particularly mediated by the complement system, stands out as a future therapeutic target in order to mitigate exacerbated BBB disruption. Locally in the lesion vicinity, additional neuroinflammatory mechanisms are in part mediated by astrocytes, where these cells seem to have an important role in local cell survival. Onwards, our findings suggest that future efforts should be directed at evaluating if neuroinflammatory modulation of complement inhibition yields improved outcome, while elaborating on the promising experimental data of astrocyte-mediated effects in the lesion vicinity

An examination of the temporal and spatial evolution of a small permanent focal ischemic lesion

The research reported here was designed to validate our hypothesis that noninvasive imaging could delineate the evolution of a small ischemic infarct. Furthermore, the alterations observed by MR were correlated to histological and inflammatory markers. Finally, intervention with a calcium buffering agent was hypothesized to prevent many of these changes. The first part of this study investigated the development of a small focal cortical lesion produced as a result of a cortical devascularization injury. Diffusion-weighted images (DWI) were collected before injury and at 12, 24, 48 hours and 3, 5, 7 and 14 days after injury and apparent diffusion coefficient (ADC) maps were calculated from the DW images to quantify lesion development. As a second measure of injury, tissue morphology was analyzed using cresyl violet histochemistry. Results indicated a significant reduction in ADC values within the lesion cortex that first appeared at 12 hours after injury and then recovered to control levels by 14 days. ADC changes were also observed in the contralateral cortex. This type of injury also resulted in the progressive but relatively slow formation of a pannecrotic infarct. Both astrocyte and microglia activation occurred early and were present in both hemispheres, however inflammatory cell infiltration was delayed until 48 hours after the injury. Many of these inflammatory cells were tumor necrosis factor a (TNF-a) and interferon y (IFN-y) immunoreactive. Overall, the quantitative and histological measures of this lesion were consistent with those observed in ischemic injury. Moreover, we found DWI to be a sensitive measure of damage associated with a cortical devascularization injury. The second part of this study used 2-aminophenol-N, N, O-triacetic acid acetoxymethyl ester (APTRA-AM) to determine the effectiveness of a calcium buffer in providing neuroprotection after a cortical devascularization injury. Animals were given two intravenous injections of either saline, DMSO, or APTRA-AM at 1 and 12 hours after injury. Animals were then imaged using a multiple b-value DWI sequence prior to injury and then at 12, 24,48 hours, 3 and 7 days after injury. After 7 days the animals were sacrificed and correlative histological and immunocytochemical studies were done. Our results indicate that saline injection after injury resulted in a decrease in the ADC of the lesion cortex within the first 12 hours of injury, which then slowly returned to prescan levels. In contrast, the injection of either DMSO or APTRA-AM after injury resulted in no significant changes in the ADC within the lesion area. Histologically, both saline and DMSO injected animals had pan-necrotic infarcts with concomitant glial activation and inflammatory cell infiltration. APTRA-AM treated animals showed an 86% reduction in lesion area and no evidence of inflammatory cell infiltration. The results presented here clearly demonstrate the effectiveness of APTRA-AM in preventing neuronal cell death and the accompanying inflammatory response when administered post-injury, suggesting that this molecule may be an excellent candidate for future clinical neuroprotection studies

Role of Astrocyte Network in Edema after Juvenile Traumatic Brain Injury

Juvenile traumatic brain injury (jTBI) is the leading cause of death and disability in young children and adolescents. Despite its lasting detrimental effects on the developing brain, no pharmacological treatment exists. One of the pathological hallmarks of jTBI is edema. Astrocytes play a key role in the edema process, and have been hypothesized that numerous astrocyte networks allow communication and propagation of edema and secondary injury spread. Two key astrocyte proteins are hypothesized to have a central role in the edema process: Aquaporin 4 (AQP4) and Connexin 43 (Cx43). AQP4 is expressed extensively in astrocyte endfeet, which surrounds the blood vessels as part of the blood brain barrier (BBB). Cx43 is central in astrocyte to astrocyte connection and communication. We hypothesized that AQP4 acted as one of the potential passageway of water into the astrocyte, whereas Cx43 acted as the bridge between astrocytes once inside the brain. By blocking these strategically located pathways, we hypothesized that edema would decrease post-jTBI. In order to achieve specific inhibitions of APQ4 or Cx43, we utilized small interference RNA (siRNA), which is also an endogenous mechanism. We observed that after jTBI both AQP4 and Cx43 was significantly upregulated, edema was prominent, and reactive astrogliosis occurred. When siAQP4 was administered after jTBI, there was functional improvement, decreased edema, and decreased reactive astrogliosis. When siCx43 was administered, there was functional improvement and decreased reactive astrogliosis, but the level of edema did not change. From these findings, it can be seen that (1) AQP4 and Cx43 are upregulated acutely after jTBI, (2) both siAQP4 and siCx43 have therapeutic potentials after jTBI leading to functional recovery, (3) although both target astrocyte endfeet proteins, the mechanism of action seem to be different and AQP4 may play a more direct role in the edema process than Cx43. Future studies could focus on (1) a more clinically relevant delivery of siRNA for jTBI, (2) elucidating the mechanism behind functional improvement of siCx43, and (3) the relationship between AQP4 and Cx43 regarding astrocyte pathology after jTBI

Relevance of Porcine Stroke Models to Bridge the Gap from Pre-Clinical Findings to Clinical Implementation

Altres ajuts: This research is supported by grants from the Fondo de Investigaciones Sanitarias-Instituto de Salud Carlos III (ISCIII) to A.D. that was susceptible to be co-financed by FEDER funds, and a grant from Agència de Gestió d'Ajuts Universitaris i de Recerca to A.D. and to T.G. The group has received funding from "la Caixa Foundation" CI15-00009, from the European Institute of Innovation and Technology (EIT), which receives support from the European Union's Horizon 2020 research and innovation programme, from the Fundación para la Innovación y la Prospectiva en Salud en España (FIPSE) program 3594-18. M.M.-S. is a recipient of a PFIS contract FI19/00174.In the search of animal stroke models providing translational advantages for biomedical research, pigs are large mammals with interesting brain characteristics and wide social acceptance. Compared to rodents, pigs have human-like highly gyrencephalic brains. In addition, increasingly through phylogeny, animals have more sophisticated white matter connectivity; thus, ratios of white-to-gray matter in humans and pigs are higher than in rodents. Swine models provide the opportunity to study the effect of stroke with emphasis on white matter damage and neuroanatomical changes in connectivity, and their pathophysiological correlate. In addition, the subarachnoid space surrounding the swine brain resembles that of humans. This allows the accumulation of blood and clots in subarachnoid hemorrhage models mimicking the clinical condition. The clot accumulation has been reported to mediate pathological mechanisms known to contribute to infarct progression and final damage in stroke patients. Importantly, swine allows trustworthy tracking of brain damage evolution using the same non-invasive multimodal imaging sequences used in the clinical practice. Moreover, several models of comorbidities and pathologies usually found in stroke patients have recently been established in swine. We review here ischemic and hemorrhagic stroke models reported so far in pigs. The advantages and limitations of each model are also discussed

Determining the temporal profile of intracranial pressure changes following transient stroke in an ovine model

Cerebral edema and elevated intracranial pressure (ICP) are the leading cause of death in the first week following stroke. Despite this, current treatments are limited and fail to address the underlying mechanisms of swelling, highlighting the need for targeted treatments. When screening promising novel agents, it is essential to use clinically relevant large animal models to increase the likelihood of successful clinical translation. As such, we sought to develop a survival model of transient middle cerebral artery occlusion (tMCAO) in the sheep and subsequently characterize the temporal profile of cerebral edema and elevated ICP following stroke in this novel, clinically relevant model. Merino-sheep (27M;31F) were anesthetized and subject to 2 h tMCAO with reperfusion or sham surgery. Following surgery, animals were allowed to recover and returned to their home pens. At preselected times points ranging from 1 to 7 days post-stroke, animals were re-anesthetized, ICP measured for 4 h, followed by imaging with MRI to determine cerebral edema, midline shift and infarct volume (FLAIR, T2 and DWI). Animals were subsequently euthanized and their brain removed for immunohistochemical analysis. Serum and cerebrospinal fluid samples were also collected and analyzed for substance P (SP) using ELISA. Intracranial pressure and MRI scans were normal in sham animals. Following stroke, ICP rose gradually over time and by 5 days was significantly (p < 0.0001) elevated above sham levels. Profound cerebral edema was observed as early as 2 days post-stroke and continued to evolve out to 6 days, resulting in significant midline shift which was most prominent at 5 days post-stroke (p < 0.01), in keeping with increasing ICP. Serum SP levels were significantly elevated (p < 0.01) by 7 days post-tMCAO. We have successfully developed a survival model of ovine tMCAO and characterized the temporal profile of ICP. Peak ICP elevation, cerebral edema and midline shift occurred at days 5-6 following stroke, accompanied by an elevation in serum SP. Our findings suggest that novel therapeutic agents screened in this model targeting cerebral edema and elevated ICP would most likely be effective when administered prior to 5 days, or as early as possible following stroke onset.Annabel J. Sorby-Adams, Anna V. Leonard, Levi E. Elms, Oana C. Marian, Jan W. Hoving, Nawaf Yassi, Robert Vink, Emma Thornton and Renée J. Turne

Caveolins and NJKs Influence Brain Endothelial Permeability after Juvenile TBI

Disruption of blood-brain barrier (BBB) is a key secondary event that exacerbates brain damage following traumatic brain injury (TBI). BBB disruption is particularly damaging to the developing brain – which is highly vulnerable to various stress stimuli, resulting in increased brain swelling, disrupted cerebral blood flow (CBF) autoregulation, long-term disabilities and death following TBI in young demographic. Unsurprisingly, BBB disruption and the resultant cerebral edema have emerged as therapeutic targets in juvenile TBI. It is therefore important to understand the molecular players and mechanisms involved in TBI-induced BBB disruption in the juvenile brain. To this end, the endothelial caveolins and c-Jun N-terminal kinases (JNKs) were identified as proteins of interest in the regulation of brain endothelial permeability after injury. These were investigated under a three-fold aim. The first was to characterize the acute and long-term histological and functional changes occurring following injury to the developing brain. Second was the attempt to profile the changes in expression patterns of caveolins after juvenile TBI in conjunction with BBB disruption. And lastly, the effects of molecular agents that target JNK (DJNKI-1) and caveolin (cavtratin) pathways respectively were examined on BBB integrity, and on imaging, histological and functional outcomes. To achieve these aims, an experimental model of TBI in juvenile rats was developed and characterized. Evidence emerged that long-term white matter dysfunction occurs in this model, in parallel with delayed neurodevelopment and persistence of behavioral deficits, which mimics data from clinical and longitudinal TBI observations. There was both acute and long-term increase in the expression level of caveolin-1 in the endothelium and reactive astrocytes following juvenile TBI. Furthermore, acute administration of cavtratin, a peptide mimetic of caveolin-1 scaffolding domain, markedly reduced edema formation and lesion volume without improving sensorimotor outcome in the acute time points. However, competitive inhibition of the JNK pathway with acute administration of DJNKI-1 markedly ameliorates BBB permeability, reduced edema formation, and improves neuroimaging and neurological outcomes at both acute and chronic time points. These findings could potentially be exploited for future therapeutic applications in juvenile brain trauma

Brain Injury

The present two volume book "Brain Injury" is distinctive in its presentation and includes a wealth of updated information on many aspects in the field of brain injury. The Book is devoted to the pathogenesis of brain injury, concepts in cerebral blood flow and metabolism, investigative approaches and monitoring of brain injured, different protective mechanisms and recovery and management approach to these individuals, functional and endocrine aspects of brain injuries, approaches to rehabilitation of brain injured and preventive aspects of traumatic brain injuries. The collective contribution from experts in brain injury research area would be successfully conveyed to the readers and readers will find this book to be a valuable guide to further develop their understanding about brain injury

The role of pericytes in microcirculatory dysfunction after subarachnoid hemorrhage

Subarachnoid hemorrhage is a subtype of stroke that is caused by a bleeding into the subarachnoid space. Cerebral ischemia develops early after the bleeding and has a negative influence on outcome in patients. The underlying pathophysiology triggering early ischemia has not been characterized in detail. Suggested pathomechnisms are pial microvasospasm, endothelial dysfunction and microthrombosis. The aim of the current thesis was to characterize and reveal the pathophysiology of microcirculatory perfusion deficits early after subarachnoid hemorrhage. The main hypothesis was that pericytes constrict upon subarachnoid hemorrhage and thereby induce capillary spasm and hamper parenchymal blood flow dynamics. We found that pial arterioles constrict in three different characteristic patterns and that spastic vessel segments were continuously covered with vascular smooth muscle cells. Superficial microvasospasm was associated with reduced blood flow velocity and significant reduction of endothelial intracellular Ca2+ concentration which may be a trigger for endothelial dysfunction. Reduced blood flow velocity in combination with reduced vessel diameter diminished total blood volume that reached the parenchymal microcirculation via penetrating arterioles. This led to a severe reduction of perfused capillary volume in the cortex. Leukocyte numbers that were sticking in capillaries and venules were increased after subarachnoid hemorrhage but their numbers were too low to explain severe perfusion deficits. Capillaries revealed a significantly reduced vessel diameter after subarachnoid hemorrhage, however vessel narrowings were not co-localizing with sites where pericytes were associated to capillaries. Furthermore pericytes neither underwent cell death nor migrated away from capillaries within 24 hours after the bleeding. In conclusion we showed that microvasospasm on the brain surface lead to severe perfusion deficits in the parenchyma. Microvasospasm are probably induced by vascular smooth muscle cells and are accompanied by reduced intracellular Ca2+ concentration in endothelial cells. Pericytes do not play a major role in the pathophysiology of early ischemia after subarachnoid hemorrhage: they neither migrate, nor die or induce capillary spasm