Characterization of Vasogenic and Cytotoxic Brain Edema Formation After Experimental Traumatic Brain Injury by Free Water Diffusion Magnetic Resonance Imaging.

Brain edema formation is a key factor for secondary tissue damage after traumatic brain injury (TBI), however, the type of brain edema and the temporal profile of edema formation are still unclear. We performed free water imaging, a bi-tensor model based diffusion MRI analysis, to characterize vasogenic brain edema (VBE) and cytotoxic edema (CBE) formation up to 7 days after experimental TBI. Male C57/Bl6 mice were subjected to controlled cortical impact (CCI) or sham surgery and investigated by MRI 4h, 1, 2, 3, 5, and 7 days thereafter (n = 8/group). We determined mean diffusivity (MD) and free water (FW) in contusion, pericontusional area, ipsi- and contralateral brain tissue. Free (i.e., non-restricted) water was interpreted as VBE, restricted water as CBE. To verify the results, VBE formation was investigated by in-vivo 2-Photon Microscopy (2-PM) 48h after surgery. We found that MD and FW values decreased for 48h within the contusion, indicating the occurrence of CBE. In pericontusional tissue, MD and FW indices were increased at all time points, suggesting the formation of VBE. This was consistent with our results obtained by 2-PM. Taken together, CBE formation occurs for 48h after trauma and is restricted to the contusion, while VBE forms in pericontusional tissue up to 7 days after TBI. Our results indicate that free water magnetic resonance imaging may represent a promising tool to investigate vasogenic and cytotoxic brain edema in the laboratory and in patients.

Balanced volatile sedation with isoflurane in critically ill patients with aneurysmal subarachnoid hemorrhage - a retrospective observational study.

Introduction: In patients with severe aneurysmal subarachnoid hemorrhage (SAH) deep sedation is often used early in the course of the disease in order to control brain edema formation and thus intracranial hypertension. However, some patients do not reach an adequate sedation depth despite high doses of common intravenous sedatives. Balanced sedation protocols incorporating low-dose volatile isoflurane administration might improve insufficient sedation depth in these patients. Methods: We retrospectively analyzed ICU patients with severe aneurysmal SAH who received isoflurane in addition to intravenous anesthetics in order to improve insufficient sedation depth. Routinely recorded data from neuromonitoring, laboratory and hemodynamic parameters were compared before and up to 6 days after initiation of isoflurane. Results: Sedation depth measured using the bispectral index improved in thirty-six SAH patients (-15.16; p = 0.005) who received additional isoflurane for a mean period of 9.73 ± 7.56 days. Initiation of isoflurane sedation caused a decline in mean arterial pressure (-4.67 mmHg; p = 0.014) and cerebral perfusion pressure (-4.21 mmHg; p = 0.013) which had to be balanced by increased doses of vasopressors. Patients required increased minute ventilation in order to adjust for the increase in PaCO2 (+2.90 mmHg; p < 0.001). We did not detect significant increases in mean intracranial pressure. However, isoflurane therapy had to be terminated prematurely in 25% of the patients after a median of 30 h due to episodes of intracranial hypertension or refractory hypercapnia. Discussion: A balanced sedation protocol including isoflurane is feasible for SAH patients experiencing inadequately shallow sedation. However, therapy should be restricted to patients without impaired lung function, hemodynamic instability and impending intracranial hypertension.

CaV2.1 channel mutations causing familial hemiplegic migraine type 1 increase the susceptibility for cortical spreading depolarizations and seizures and worsen outcome after experimental traumatic brain injury.

Patients suffering from familial hemiplegic migraine type 1 (FHM1) may have a disproportionally severe outcome after head trauma, but the underlying mechanisms are unclear. Hence, we subjected knock-in mice carrying the severer S218L or milder R192Q FHM1 gain-of-function missense mutation in the CACNA1A gene that encodes the α1A subunit of neuronal voltage-gated CaV2.1 (P/Q-type) calcium channels and their wild-type (WT) littermates to experimental traumatic brain injury (TBI) by controlled cortical impact and investigated cortical spreading depolarizations (CSDs), lesion volume, brain edema formation, and functional outcome. After TBI, all mutant mice displayed considerably more CSDs and seizures than WT mice, while S218L mutant mice had a substantially higher mortality. Brain edema formation and the resulting increase in intracranial pressure were more pronounced in mutant mice, while only S218L mutant mice had larger lesion volumes and worse functional outcome. Here, we show that gain of CaV2.1 channel function worsens histopathological and functional outcome after TBI in mice. This phenotype was associated with a higher number of CSDs, increased seizure activity, and more pronounced brain edema formation. Hence, our results suggest increased susceptibility for CSDs and seizures as potential mechanisms for bad outcome after TBI in FHM1 mutation carriers.

Perfusion pressure determines vascular integrity and histomorphological quality following perfusion fixation of the brain.

INTRODUCTION: Histology on fixed brain tissue is a key technique to investigate the pathophysiology of neurological disorders. Best results are obtained by perfusion fixation, however, multiple protocols are available and so far the optimal perfusion pressure (PP) for the preservation of brain tissue while also maintaining vascular integrity is not defined. Therefore, the aim of our study was to investigate the effect of different PPs on the cerebral vasculature and to define the PP optimal for the preservation of both vascular integrity and tissue fixation. MATERIAL AND METHODS: Male C57Bl6 mice, 8 weeks old, were perfused with PPs of 50/125/300 mmHg (series I) or 50/100/150/300 mmHg (series II). In series I, vascular integrity, e.g. BBB permeability, vessel diameter, and occurrence of vasospasms were investigated by spectrophotometry, light-sheet and 2-photon microscopy, respectively. In series II, we investigated vascular and neuronal artifacts and the occurrence of hemorrhage or microthrombi by light microscopy. RESULTS: While a PP below the physiological systolic blood pressure results in the collapse of parenchymal vessels and formation of microvasospasms and microclots, a PP above the physiological systolic blood pressure dilates cerebral vessels, induces microvasospasms and disrupts the BBB. In terms of tissue integrity, our results confirm that higher PPs lead to fewer artifacts such as dark neurons or perivascular courts. CONCLUSION: Our study demonstrates that the PP critically affects both vascular and tissue integrity in brain tissue preserved by perfusion fixation. A PP between 125 and 150 mmHg is optimal for the preservation of the cerebral vasculature and neuronal structures.

Longitudinal Characterization of Blood-Brain Barrier Permeability after Experimental Traumatic Brain Injury by In Vivo 2-Photon Microscopy.

Vasogenic brain edema (VBE) formation remains an important factor determining the fate of patients with traumatic brain injury (TBI). The spatial and temporal development of VBE, however, remains poorly understood because of the lack of sufficiently sensitive measurement techniques. To close this knowledge gap, we directly visualized the full time course of vascular leakage after TBI by in vivo 2-photon microscopy (2-PM). Male C57BL/6 mice (n = 6/group, 6-8 weeks old) were assigned randomly to sham operation or brain trauma by controlled cortical impact. A cranial window was prepared, and tetramethylrhodamine-dextran (TMRM, MW 40,000 Da) was injected intravenously to visualize blood plasma 4 h, 24 h, 48 h, 72 h, or seven days after surgery or trauma. Three regions with increasing distance to the primary contusion were investigated up to a depth of 300 µm by 2-PM. No TMRM extravasation was detected in sham-operated mice, while already 4 h after TBI vascular leakage was significantly increased (p < 0.05 vs. sham) and reached its maximum at 48 h after injury. Vascular leakage was most pronounced in the vicinity of the contusion. The rate of extravasation showed a biphasic pattern, peaking 4 h and 48-72 h after trauma. Taken together, longitudinal quantification of vascular leakage after TBI in vivo demonstrates that VBE formation after TBI develops in a biphasic manner suggestive of acute and delayed mechanisms. Further studies using the currently developed dynamic in vivo imaging modalities are needed to investigate these mechanisms and potential therapeutic strategies in more detail.

Plasminogen activator inhibitor-1 augments damage by impairing fibrinolysis after traumatic brain injury.

OBJECTIVE: Plasminogen activator inhibitor-1 (PAI-1) is the key endogenous inhibitor of fibrinolysis, and enhances clot formation after injury. In traumatic brain injury, dysregulation of fibrinolysis may lead to sustained microthrombosis and accelerated lesion expansion. In the present study, we hypothesized that PAI-1 mediates post-traumatic malfunction of coagulation, with inhibition or genetic depletion of PAI-1 attenuating clot formation and lesion expansion after brain trauma. METHODS: We evaluated PAI-1 as a possible new target in a mouse controlled cortical impact (CCI) model of traumatic brain injury. We performed the pharmacological inhibition of PAI-1 with PAI-039 and stimulation by tranexamic acid, and we confirmed our results in PAI-1-deficient animals. RESULTS: PAI-1 mRNA was time-dependently upregulated, with a 305-fold peak 12 hours after CCI, which effectively counteracted the 2- to 3-fold increase in cerebral tissue-type/urokinase plasminogen activator expression. PAI-039 reduced brain lesion volume by 26% at 24 hours and 43% at 5 days after insult. This treatment also attenuated neuronal apoptosis and improved neurofunctional outcome. Moreover, intravital microscopy demonstrated reduced post-traumatic thrombus formation in the pericontusional cortical microvasculature. In PAI-1-deficient mice, the therapeutic effect of PAI-039 was absent. These mice also displayed 13% reduced brain damage compared with wild type. In contrast, inhibition of fibrinolysis with tranexamic acid increased lesion volume by 25% compared with vehicle. INTERPRETATION: This study identifies impaired fibrinolysis as a critical process in post-traumatic secondary brain damage and suggests that PAI-1 may be a central endogenous inhibitor of the fibrinolytic pathway, promoting a procoagulatory state and clot formation in the cerebral microvasculature. Ann Neurol 2019;85:667-680.

Microvasospasms After Experimental Subarachnoid Hemorrhage Do Not Depend on Endothelin A Receptors.

BACKGROUND AND PURPOSE: Perturbations in cerebral microcirculation (eg, microvasospasms) and reduced neurovascular communication determine outcome after subarachnoid hemorrhage (SAH). ET-1 (endothelin-1) and its receptors have been implicated in the pathophysiology of large artery spasms after SAH; however, their role in the development of microvascular dysfunction is currently unknown. Here, we investigated whether inhibiting ETA (endothelin A) receptors can reduce microvasospasms after experimentally induced SAH. METHODS: SAH was induced in male C57BL/6 mice by filament perforation of the middle cerebral artery. Three hours after SAH, a cranial window was prepared and the pial and parenchymal cerebral microcirculation was measured in vivo using two-photon microscopy before, during, and after administration of the ETA receptor inhibitor clazosentan. In separate experiments, the effect of clazosentan treatment on neurological outcome was measured 3 days after SAH. RESULTS: Clazosentan treatment had no effect on the number or severity of SAH-induced cerebral microvasospasms nor did it affect neurological outcome. CONCLUSIONS: Our results indicate that ETA receptors, which mediate large artery spasms after SAH, do not seem to play a role in the development of microarterial spasms, suggesting that posthemorrhagic spasms are mediated by distinct mechanisms in large and small cerebral vessels. Given that cerebral microvessel dysfunction is a key factor for outcome after SAH, further research into the mechanisms that underlie posthemorrhagic microvasospasms is urgently needed.

Acute changes in neurovascular reactivity after subarachnoid hemorrhage in vivo.

Subarachnoid hemorrhage causes acute and long-lasting constrictions of pial arterioles. Whether these vessels dilate normally to neuronal activity is of great interest since a mismatch between delivery and consumption of glucose and oxygen may cause additional neuronal damage. Therefore, we investigated neurovascular reactivity of pial and parenchymal arterioles after experimental subarachnoid hemorrhage. C57BL/6 mice were subjected to subarachnoid hemorrhage by filament perforation or sham surgery. Neurovascular reactivity was assessed 3 h later by forepaw stimulation or inhalation of 10% CO2 Diameters of cerebral arterioles were assessed using two-photon microscopy. Neurovascular coupling and astrocytic endfoot Ca2+ were measured in brain slices using two-photon and infrared-differential interference contrast microscopy. Vessels of sham-operated mice dilated normally to CO2 and forepaw stimulation. Three hours after subarachnoid hemorrhage, CO2 reactivity was completely lost in both pial and parenchymal arterioles, while neurovascular coupling was not affected. Brain slices studies also showed normal neurovascular coupling and a normal increase in astrocytic endfoot Ca2+ acutely after subarachnoid hemorrhage. These findings suggest that communication between neurons, astrocytes, and parenchymal arterioles is not affected in the first few hours after subarachnoid hemorrhage, while CO2 reactivity, which is dependent on NO signaling, is completely lost.

The Formation of Microthrombi in Parenchymal Microvessels after Traumatic Brain Injury Is Independent of Coagulation Factor XI.

Microthrombus formation and bleeding worsen the outcome after traumatic brain injury (TBI). The aim of the current study was to characterize these processes in the brain parenchyma after experimental TBI and to determine the involvement of coagulation factor XI (FXI). C57BL/6 mice (n = 101) and FXI-deficient mice (n = 15) were subjected to controlled cortical impact (CCI). Wild-type mice received an inhibitory antibody against FXI (14E11) or control immunoglobulin G 24 h before or 30 or 120 min after CCI. Cerebral microcirculation was visualized in vivo by 2-photon microscopy 2-3 h post-trauma and histopathological outcome was assessed after 24 h. TBI induced hemorrhage and microthrombus formation in the brain parenchyma (p < 0.001). Inhibition of FXI activation or FXI deficiency did not reduce cerebral thrombogenesis, lesion volume, or hemispheric swelling. However, it also did not increase intracranial hemorrhage. Formation of microthrombosis in the brain parenchyma after TBI is independent of the intrinsic coagulation cascade since it was not reduced by inhibition of FXI. However, since targeting FXI has well-established antithrombotic effects in humans and experimental animals, inhibition of FXI could represent a reasonable strategy for the prevention of deep venous thrombosis in immobilized patients with TBI.

Applying the retro-enantio approach to obtain a peptide capable of overcoming the blood-brain barrier.

The blood-brain barrier (BBB) is a formidable physical and enzymatic barrier that tightly controls the passage of molecules from the blood to the brain. In fact, less than 2 % of all potential neurotherapeutics are able to cross it. Here, by applying the retro-enantio approach to a peptide that targets the transferrin receptor, a full protease-resistant peptide with the capacity to act as a BBB shuttle was obtained and thus enabled the transport of a variety of cargos into the central nervous system.

Identification of the Vascular Source of Vasogenic Brain Edema following Traumatic Brain Injury Using In Vivo 2-Photon Microscopy in Mice.

Vasogenic brain edema due to vascular leakage is one of the most important factors determining the clinical outcome of patients following acute brain injury. To date, performing a detailed in vivo quantification of vascular leakage has not been possible. Here, we used in vivo 2-photon microscopy (2-PM) to determine the spatial (3D) and temporal development of vasogenic brain edema following traumatic brain injury (TBI) in mice; in addition, we identified the vessel types involved in vascular leakage. Thirteen male Tie2-GFP mice (6-8 weeks old) were subjected to controlled cortical impact (CCI) or a sham operation; subsequently, a cranial window was prepared adjacent to the injury site, and tetramethylrhodamine-dextran (TMRM, 40 mg/kg, MW 40,000) was injected intravenously to visualize blood plasma leakage. Parenchymal fluorescence intensity was monitored in three regions for 2-4 h post-CCI, reaching from the surface of the brain to a depth of 300 µm, and TMRM leakage was measured as an increase in TMRM fluorescence intensity outside the vessel lumen and in the parenchyma. In the CCI group, vascular leakage was detected in all investigated regions as early as 2.5 h post-injury. This leakage increased over time and was more pronounced proximal to the primary contusion. Both arterioles and venules contributed similarly to brain edema formation and their contribution was independent of vessel size; however, capillaries were the major contributor to leakage. In summary, using 2-PM to perform in vivo 3D deep-brain imaging, we found that TBI induces vascular leakage from capillaries, venules, and arterioles. Thus, all three vessel types are involved in trauma-induced brain edema and should be considered when developing novel therapies for preventing vasogenic brain edema.

Endothelial nitric oxide synthase mediates arteriolar vasodilatation after traumatic brain injury in mice.

Brain edema and increased cerebral blood volume (CBV) contribute to intracranial hypertension and hence to unfavorable outcome after traumatic brain injury (TBI). The increased post-traumatic CBV may be caused in part by arterial vasodilatation. The aim of the current study was to uncover the largely unknown mechanisms of post-traumatic arteriolar vasodilatation. The diameter of pial arterioles and venules was monitored by intravital fluorescence microscopy before (baseline) and for 30 min after controlled cortical impact in C57BL/6 and endothelial nitric oxide synthase (eNOS)-/- mice (n=5-6/group) and in C57BL/6 mice (n=6/group) receiving vehicle (phosphate-buffered saline [PBS]) or 4-amino-tetrahydro-L-biopterine (VAS203), a NOS inhibitor previously shown to reduce post-traumatic intracranial hypertension. Temperature, end-tidal partial pressure of carbon dioxide (pCO2), and mean arterial blood pressure were kept within the physiological range throughout the experiments. Arteriolar diameters were stable during baseline monitoring but increased significantly in C57BL/6 mice after controlled cortical impact (136±7% of baseline; p<0.001 vs. baseline). This response was reduced by 78% in eNOS-/- mice (108±3% of baseline; p<0.005 vs. wild-type). Application of VAS203, a NOS inhibitor, or PBS did not affect vessels diameter before TBI. After trauma, however, administration of VAS203 reduced arteriolar diameter to 92±2% of baseline (p<0.05). The diameter of pial veins was not affected. Our results suggest that arteriolar vasodilatation after TBI is largely mediated by excess production of endothelial nitric oxide. Accordingly, our data may explain the beneficial effects of the NOS inhibitor VAS203 in the early phase after TBI and suggest that inhibition of excess endothelial nitric oxide production may represent a novel therapeutic strategy following TBI.

Contributions of the immune system to the pathophysiology of traumatic brain injury - evidence by intravital microscopy.

Traumatic brain injury (TBI) results in immediate brain damage that is caused by the mechanical impact and is non-reversible. This initiates a cascade of delayed processes which cause additional-secondary-brain damage. Among these secondary mechanisms, the inflammatory response is believed to play an important role, mediating actions that can have both protective and detrimental effects on the progression of secondary brain damage. Histological data generated extensive information; however, this is only a snapshot of processes that are, in fact, very dynamic. In contrast, in vivo microscopy provides detailed insight into the temporal and spatial patterns of cellular dynamics. In this review, we aim to summarize data which was generated by in vivo microscopy, specifically investigating the immune response following brain trauma, and its potential effects on secondary brain damage.

In vivo temporal and spatial profile of leukocyte adhesion and migration after experimental traumatic brain injury in mice.

BACKGROUND: Leukocytes are believed to be involved in delayed cell death following traumatic brain injury (TBI). However, data demonstrating that blood-borne inflammatory cells are present in the injured brain prior to the onset of secondary brain damage have been inconclusive. We therefore investigated both the interaction between leukocytes and the cerebrovascular endothelium using in vivo imaging and the accumulation of leukocytes in the penumbra following experimentally induced TBI. METHODS: Experimental TBI was induced in C57/Bl6 mice (n = 42) using the controlled cortical impact (CCI) injury model, and leukocyte-endothelium interactions (LEI) were quantified using both intravital fluorescence microscopy (IVM) of superficial vessels and 2-photon microscopy of cortical vessels for up to 14 h post-CCI. In a separate experimental group, leukocyte accumulation and secondary lesion expansion were analyzed in mice that were sacrificed 15 min, 2, 6, 12, 24, or 48 h after CCI (n = 48). Finally, leukocyte adhesion was blocked with anti-CD18 antibodies, and the effects on LEI and secondary lesion expansion were determined 16 (n = 12) and 24 h (n = 21), respectively, following TBI. RESULTS: One hour after TBI leukocytes and leukocyte-platelet aggregates started to roll on the endothelium of pial venules, whereas no significant LEI were observed in pial arterioles or in sham-operated mice. With a delay of >4 h, leukocytes and aggregates did also firmly adhere to the venular endothelium. In deep cortical vessels (250 µm) LEIs were much less pronounced. Transmigration of leukocytes into the brain parenchyma only became significant after the tissue became necrotic. Treatment with anti-CD18 antibodies reduced adhesion by 65%; however, this treatment had no effect on secondary lesion expansion. CONCLUSIONS: LEI occurred primarily in pial venules, whereas little or no LEI occurred in arterioles or deep cortical vessels. Inhibiting LEI did not affect secondary lesion expansion. Importantly, the majority of migrating leukocytes entered the injured brain parenchyma only after the tissue became necrotic. Our results therefore suggest that neither intravascular leukocyte adhesion nor the migration of leukocytes into cerebral tissue play a significant role in the development of secondary lesion expansion following TBI.

Temporal profile of thrombogenesis in the cerebral microcirculation after traumatic brain injury in mice.

Traumatic brain injury (TBI) is associated with an almost immediate reduction in cerebral blood flow (CBF). Because cerebral perfusion pressure is often normal under these circumstances it was hypothesized that the reduction of post-traumatic CBF has to occur at the level of the microcirculation. The aim of the current study was to investigate whether cerebral microvessels are involved in the development of blood flow disturbances following experimental TBI. C57/BL6 mice (n = 12) were intubated and ventilated under control of end-tidal Pco(2) ((ET)P(CO2)). After preparation of a cranial window and baseline recordings, the animals were subjected to experimental TBI by controlled cortical impact (CCI; 6 m/sec, 0.5 mm). Vessel lumina and intravascular cells were visualized by in vivo fluorescence microscopy (IVM) using the fluorescent dyes FITC-dextran and rhodamine 6G, respectively. Vessel diameter, cell-endothelial interactions, and thrombus formation were quantified within the traumatic penumbra by IVM up to 2 h after CCI. Arteriolar diameters increased after CCI by 26.2 +/- 2.5% (mean +/- SEM, p < 0.01 versus baseline), and remained at this level until the end of the observation period. Rolling of leukocytes on the cerebrovascular endothelium was observed both in arterioles and venules, while leukocyte-platelet aggregates were found only in venules. Microthrombi occluded up to 70% of venules and 33% of arterioles. The current data suggest that the immediate post-traumatic decrease in peri-contusional blood flow is not caused by arteriolar vasoconstriction, but by platelet activation and the subsequent formation of thrombi in the cerebral microcirculation.