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

Neurovascular Reactivity in the Aging Mouse Brain Assessed by Laser Speckle Contrast Imaging and 2-Photon Microscopy: Quantification by an Investigator-Independent Analysis Tool

The brain has a high energy demand but little to no energy stores. Therefore, proper brain function relies on the delivery of glucose and oxygen by the cerebral vasculature. The regulation of cerebral blood flow (CBF) occurs at the level of the cerebral capillaries and is driven by a fast and efficient crosstalk between neurons and vessels, a process termed neurovascular coupling (NVC). Experimentally NVC is mainly triggered by sensory stimulation and assessed by measuring either CBF by laser Doppler fluxmetry, laser speckle contrast imaging (LSCI), intrinsic optical imaging, BOLD fMRI, near infrared spectroscopy (NIRS) or functional ultrasound imaging (fUS). Since these techniques have relatively low spatial resolution, diameters of cerebral vessels are mainly assessed by 2-photon microscopy (2-PM). Results of studies on NVC rely on stable animal physiology, high-quality data acquisition, and unbiased data analysis, criteria, which are not easy to achieve. In the current study, we assessed NVC using two different imaging modalities, i.e., LSCI and 2-PM, and analyzed our data using an investigator-independent Matlab-based analysis tool, after manually defining the area of analysis in LSCI and vessels to measure in 2-PM. By investigating NVC in 6–8 weeks, 1-, and 2-year-old mice, we found that NVC was maximal in 1-year old mice and was significantly reduced in aged mice. These findings suggest that NVC is differently affected during the aging process. Most interestingly, specifically pial arterioles, seem to be distinctly affected by the aging. The main finding of our study is that the automated analysis tool works very efficiently in terms of time and accuracy. In fact, the tool reduces the analysis time of one animal from approximately 23 h to about 2 s while basically making no mistakes. In summary, we developed an experimental workflow, which allows us to reliably measure NVC with high spatial and temporal resolution in young and aged mice and to analyze these data in an investigator-independent manner

Ultrabright Fluorescent Polymeric Nanoparticles with a Stealth Pluronic Shell for Live Tracking in the Mouse Brain

Visualizing single organic nanoparticles (NPs) in vivo remains a challenge, which could greatly improve our understanding of the bottlenecks in the field of nanomedicine. To achieve high single-particle fluorescence brightness, we loaded polymer poly(methyl methacrylate)-sulfonate (PMMA-SO3H) NPs with octadecyl rhodamine B together with a bulky hydrophobic counterion (perfluorinated tetraphenylborate) as a fluorophore insulator to prevent aggregation-caused quenching. To create NPs with stealth properties, we used the amphiphilic block copolymers pluronic F-127 and F-68. Fluorescence correlation spectroscopy and Förster resonance energy transfer (FRET) revealed that pluronics remained at the NP surface after dialysis (at one amphiphile per 5.5 nm2) and prevented NPs from nonspecific interactions with serum proteins and surfactants. In primary cultured neurons, pluronics stabilized the NPs, preventing their prompt aggregation and binding to neurons. By increasing dye loading to 20 wt % and optimizing particle size, we obtained 74 nm NPs showing 150-fold higher single-particle brightness with two-photon excitation than commercial Nile Red-loaded FluoSpheres of 39 nm hydrodynamic diameter. The obtained ultrabright pluronic-coated NPs enabled direct single-particle tracking in vessels of mice brains by two-photon intravital microscopy for at least 1 h, whereas noncoated NPs were rapidly eliminated from the circulation. Following brain injury or neuroinflammation, which can open the blood–brain barrier, extravasation of NPs was successfully monitored. Moreover, we demonstrated tracking of individual NPs from meningeal vessels until their uptake by meningeal macrophages. Thus, single NPs can be tracked in animals in real time in vivo in different brain compartments and their dynamics visualized with subcellular resolution

Inversion of neurovascular coupling after subarachnoid hemorrhage in vivo

Subarachnoid hemorrhage (SAH) induces acute changes in the cerebral microcirculation. Recent findings ex vivo suggest neurovascular coupling (NVC), the process that increases cerebral blood flow upon neuronal activity, is also impaired after SAH. The aim of the current study was to investigate whether this occurs also invivo. C57BL/6 mice were subjected to either sham surgery or SAH by filament perforation. Twenty-four hours later NVC was tested by forepaw stimulation and CO2 reactivity by inhalation of 10% CO2. Vessel diameter was assessed invivo by two-photon microscopy. NVC was also investigated ex vivo using brain slices. Cerebral arterioles of sham-operated mice dilated to 130% of baseline upon CO2 inhalation or forepaw stimulation and cerebral blood flow (CBF) increased. Following SAH, however, CO2 reactivity was completely lost and the majority of cerebral arterioles showed paradoxical constriction invivo and ex vivo resulting in a reduced CBF response. As previous results showed intact NVC 3h after SAH, the current findings indicate that impairment of NVC after cerebral hemorrhage occurs secondarily and is progressive. Since neuronal activity-induced vasoconstriction (inverse NVC) is likely to further aggravate SAH-induced cerebral ischemia and subsequent brain damage, inverse NVC may represent a novel therapeutic target after SAH

Function of BID - a molecule of the bcl-2 family - in ischemic cell death in the brain

Mitochondrial mechanisms, particularly the release of cytochrome c, play a role in the death of nerve and glial cells in cerebral ischemia. We have currently investigated whether BID, a proapoptotic molecule of the bcl-2 family and promoter of the release of cytochrome c is expressed in the brain, activated by cerebral ischemia in vivo, and contributes to ischemic cell death. We found BID in the cytosol of mouse brain and of primary cultured mouse neurons and showed that neuronal BID is a substrate for caspase 8. BID was cleaved in vivo 4 h after transitory occlusion of the middle cerebral artery. Further, BID-/- mice had a significant attenuation of infarction (-67%) and significantly lower release of cytochrome c (-41 %). The findings indicate that the proapoptotic molecule BID may contribute to the demise of nerve cells from cerebral ischemia by release of cytochrome c and activation of caspase. Copyright (C) 2002 S. Karger AG, Basel

Adhesion of Leukocytes to Cerebral Venules Precedes Neuronal Cell Death and Is Sufficient to Trigger Tissue Damage After Cerebral Ischemia

BackgroundLeukocytes contribute to tissue damage after cerebral ischemia;however, the mechanisms underlying this process are still unclear. This study investigates the temporal and spatial relationship between vascular leukocyte recruitment and tissue damage and aims to uncover which step of the leukocyte recruitment cascade is involved in ischemic brain injury. MethodsMale wild-type, ICAM-1-deficient, anti-CD18 antibody treated, or selectin-deficient [fucusyltransferase (FucT IV/VII-/-)] mice were subjected to 60 min of middle cerebral artery occlusion (MCAo). The interaction between leukocytes and the cerebrovascular endothelium was quantified by in vivo fluorescence microscopy up to 15 h thereafter. Temporal dynamics of neuronal cell death and leukocyte migration were assessed at the same time points and in the same tissue volume by histology. ResultsIn wild-type mice, leukocytes started to firmly adhere to the wall of pial postcapillary venules two hours after reperfusion. Three hours later, neuronal loss started and 13 h later, leukocytes transmigrated into brain tissue. Loss of selectin function did not influence this process. Application of an anti-CD18 antibody or genetic deletion of ICAM-1, however, significantly reduced tight adhesion of leukocytes to the cerebrovascular endothelium (-60%;p < 0.01) and increased the number of viable neurons in the ischemic penumbra by 5-fold (p < 0.01);the number of intraparenchymal leukocytes was not affected. ConclusionsOur findings suggest that ischemia triggers only a transient adhesion of leukocytes to the venous endothelium and that inhibition of this process is sufficient to partly prevent ischemic tissue damage

Decompressive Craniectomy Is Associated With Good Quality of Life Up to 10 Years After Rehabilitation From Traumatic Brain Injury

OBJECTIVES Traumatic brain injury is the number one cause of death in children and young adults and has become increasingly prevalent in the elderly. Decompressive craniectomy prevents intracranial hypertension but does not clearly improve physical outcome 6 months after traumatic brain injury. However, it has not been analyzed if decompressive craniectomy affects traumatic brain injury patients' quality of life in the long term. DESIGN Therefore, we conducted a cross-sectional study assessing health-related quality of life in traumatic brain injury patients with or without decompressive craniectomy up to 10 years after injury. SETTING Former critical care patients. PATIENTS Chronic traumatic brain injury patients having not (n = 37) or having received (n = 98) decompressive craniectomy during the acute treatment. MEASUREMENTS AND MAIN RESULTS The Quality of Life after Brain Injury questionnaire was used as outcome measure with a total score from zero to 100, representing lowest and best health-related quality of life, respectively. Health-related quality of life was compared between patients with or without decompressive craniectomy for the entire cohort, for the traumatic brain injury severity (mild, moderate, severe) measured by the initial Glasgow Coma Scale, for age and time variables (age at traumatic brain injury, age at survey, elapsed time since traumatic brain injury) using the Mann-Whitney U test. Differences were considered significant at a p value of less than 0.05.Decompressive craniectomy was necessary in all initial traumatic brain injury severity groups. Eight percent more decompressive craniectomy patients reported good health-related quality of life with a Quality of Life after Brain Injury total score greater than or equal to 60 compared with the no decompressive craniectomy patients up to 10 years after traumatic brain injury (p = 0.004). Initially, mild classified traumatic brain injury patients had a median Quality of Life after Brain Injury total score of 83 (decompressive craniectomy) versus 62 (no decompressive craniectomy) (p = 0.028). Health-related quality of life regarding physical status was better in decompressive craniectomy patients (p = 0.025). Decompressive craniectomy showed a trend toward better health-related quality of life in the 61-85-year-old reflected by median Quality of Life after Brain Injury total scores of 62 (no decompressive craniectomy) versus 79 (decompressive craniectomy) (p = 0.06). CONCLUSIONS Our results suggest that decompressive craniectomy is associated with good health-related quality of life up to 10 years after traumatic brain injury. Thus, decompressive craniectomy may have an underestimated therapeutic potential after traumatic brain injury

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 &gt;4 h, leukocytes and aggregates did also firmly adhere to the venular endothelium. In deep cortical vessels (250 mu m) 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