Fabrizio Ortolano

CX3CR1 deficiency induces an early protective inflammatory environment in ischemic mice

The studies on fractalkine and its unique receptor CX3CR1 in neurological disorders yielded contrasting results. We have explored the consequences of CX3CR1 deletion in ischemic (30′ MCAo) mice on: (1) brain infarct size; (2) microglia dynamism and morphology; (3) expression of markers of microglia/macrophages (M/M) activation and polarization. We observed smaller infarcts in cx3cr1−/− (26.42 ± 7.41 mm3, mean ± sd) compared to wild type (36.29 ± 11.57) and cx3cr1−/+ (34.49 ± 8.91) mice. We longitudinally analyzed microglia by in vivo two‐photon microscopy before, 1 and 24 h after transient ischemia. Microglia were stationary in both cx3cr1−/− and cx3cr1−/+ mice throughout the study. In cx3cr1−/− mice, they displayed a significantly higher number of ramifications >10 μm at baseline and at 24 h after ischemia compared to cx3cr1−/+ mice, indicating that CX3CR1 deficiency impaired the development of microglia hypertrophic/amoeboid morphology. At 24 h after ischemia, we performed post mortem quantitative immunohistochemistry for different M/M markers. In cx3cr1−/− immunoreactivity for CD11b (M/M activation) and for CD68 (associated with phagocytosis) were decreased, while that for CD45high (macrophage and leukocyte recruitment) was increased. In addition, immunoreactivity for Ym1 (M2 polarization) was enhanced, while that for iNOS (M1) was decreased. Our data show that in cx3cr1−/− mice protection from ischemia at early time points after injury is associated with a protective inflammatory milieu, characterized by the promotion of M2 polarization markers.

Long-lasting protection in brain trauma by endotoxin preconditioning

We investigated the occurrence of endotoxin (lipopolysaccharide, LPS) preconditioning in traumatic brain injury (TBI), evaluating the time window of LPS-induced protection, its persistence, and the associated molecular mechanisms. Mice received 0.1 mg/kg LPS or saline intraperitoneally and subsequently TBI (by controlled cortical impact brain injury) at various time intervals. Mice receiving LPS 3, 5, or 7 days before TBI showed attenuated motor deficits at 1 week after injury compared with mice receiving saline. Those receiving LPS 5 days before injury had also a reduced contusion volume (7.9 ± 1.3 versus 12 ± 2.3 mm3) and decreased cell death. One month after injury, the protective effect of LPS on contusion volume (14.5 ± 1.2 versus 18.2 ± 1.2 mm3) and neurologic function was still present. Traumatic brain injury increased glial fibrillary acidic protein, CD11b, CD68, tumor necrosis factor-α, interleukin (IL)-10, and IL-6 mRNA expression 24 hours after injury. Lipopolysaccharide administered 5 (but not 9) days before injury increased the expression of CD11b (233%) and of interferon β (500%) in uninjured mice, while it reduced the expression of CD68 (by 46%) and increased that of IL-6 (by 52%) in injured mice. Lipopolysaccharide preconditioning conferred a long-lasting neuroprotection after TBI, which was associated with a modulation of microglia/macrophages activity and cytokine production.

Advances in imaging of new targets for pharmacological intervention in stroke: real-time tracking of T-cells in the ischaemic brain

Background and purpose:  T‐cells may play a role in the evolution of ischaemic damage and repair, but the ability to image these cells in the living brain after a stroke has been limited. We aim to extend the technique of real‐time in situ brain imaging of T‐cells, previously shown in models of immunological diseases, to models of experimental stroke.

Neuroprotective effect of C1-inhibitor following traumatic brain injury in mice

BACKGROUND The goal of the study was to evaluate the effects of Cl-inhibitor (C1-INH), an endogenous glycoprotein endowed with multiple anti-inflammatory actions, on cognitive and histological outcome following controlled cortical impact (CCI) brain injury. METHODS Male C57B1/6 mice (n=48) were subjected to CCI brain injury. After brain injury, animals randomly received an intravenous infusion of either C1-INH (15 U either at 10 minutes or 1 hour postinjury) or saline (equal volume, 150 microl at 10 min postinjury). Uninjured control mice received identical surgery and saline injection without brain injury. Cognitive function was evaluated at 4 weeks postinjury using the Morris Water Maze. Mice were subsequently sacrificed, the brains were frozen and serial sections were cut. Traumatic brain lesion was assessed by dividing the area of the ipsilateral hemisphere for the area of the contralateral one at the level of the injured area of the brain. FINDINGS Brain-injured mice receiving C1-INH at 10 min postinjury showed attenuated cognitive dysfunction compared to brain-injured mice receiving saline (p < 0.01). These mice also showed a significantly reduced traumatic brain lesion compared to mice receiving saline (p < 0.01). Mice receiving C1-INH at 1 hour post injury did not show a significant improvement in either cognitive or histological outcome. Conclusions Our results suggest that administration of C1-INH at 10 minutes postinjury attenuates cognitive deficits and histological damage associated with traumatic brain injury.

Imaging T‐cell movement in the brain during experimental cerebral malaria

T‐cells are known to play a role in the pathology associated with experimental cerebral malaria, although it has not previously been possible to examine their behaviour in brain. Using multiphoton laser scanning microscopy, we have examined the migration and movement of these cells in brain tissue. We believe that this approach will help define host–parasite interactions and examine how intervening in these relationships affects the development of cerebral pathology.

A close look at brain dynamics: cells and vessels seen by in vivo two-photon microscopy.

The cerebral vasculature has a unique role in providing a constant supply of oxygen and nutrients to ensure normal brain functions. Blood vessels that feed the brain are far from being simply channels for passive transportation of fluids. They form complex structures made up of different cell types. These structures regulate blood supply, local concentrations of O2 and CO2, transport of small molecules, trafficking of plasma cells and fine cerebral functions in normal and diseased brains. Until few years ago, analysis of these functions has been typically based on post mortem techniques, whose interpretation is limited by the need for tissue processing at specific times. For a reliable and effective picture of the dynamic processes in the central nervous system, real-time information in vivo is required. There are now few in vivo systems, among which two-photon microscopy (2-PM) is a truly innovative tool for studying the brain. 2-PM has been used to dissect specific aspects of vascular and immune cell dynamics in the context of neurological diseases, providing exciting results that could not have been obtained with conventional methods. This review summarizes the latest findings on vascular and immune system action in the brain, with particular focus on the dynamic responses after ischemic brain injury. 2-PM has helped define the hierarchical architecture of the brain vasculature, the dynamic interaction between the vasculature and immune cells recruited to lesion sites, the effects of blood flow on neuronal and microglial activity and the ability of cells of the neurovascular unit to regulate blood flow.

Rethinking Neuroprotection in Severe Traumatic Brain Injury: Toward Bedside Neuroprotection

Neuroprotection after traumatic brain injury (TBI) is an important goal pursued strenuously in the last 30 years. The acute cerebral injury triggers a cascade of biochemical events that may worsen the integrity, function, and connectivity of the brain cells and decrease the chance of functional recovery. A number of molecules acting against this deleterious cascade have been tested in the experimental setting, often with preliminary encouraging results. Unfortunately, clinical trials using those candidate neuroprotectants molecules have consistently produced disappointing results, highlighting the necessity of improving the research standards. Despite repeated failures in pharmacological neuroprotection, TBI treatment in neurointensive care units has achieved outcome improvement. It is likely that intensive treatment has contributed to this progress offering a different kind of neuroprotection, based on a careful prevention and limitations of intracranial and systemic threats. The natural course of acute brain damage, in fact, is often complicated by additional adverse events, like the development of intracranial hypertension, brain hypoxia, or hypoperfusion. All these events may lead to additional brain damage and worsen outcome. An approach designed for early identification and prompt correction of insults may, therefore, limit brain damage and improve results.

Human brain trauma severity is associated with lectin complement pathway activation

We explored the involvement of the lectin pathway of complement in post-traumatic brain injury (TBI) pathophysiology in humans. Brain samples were obtained from 28 patients who had undergone therapeutic contusion removal, within 12 h (early) or from >12 h until five days (late) from injury, and from five non-TBI patients. Imaging analysis indicated that lectin pathway initiator molecules (MBL, ficolin-1, ficolin-2 and ficolin-3), the key enzymes MASP-2 and MASP-3, and the downstream complement components (C3 fragments and TCC) were present inside and outside brain vessels in all contusions. Only ficolin-1 was found in the parenchyma of non-TBI tissues. Immunoassays in brain homogenates showed that MBL, ficolin-2 and ficolin-3 increased in TBI compared to non-TBI (2.0, 2.2 and 6.0-times) samples. MASP-2 increased with subarachnoid hemorrhage and abnormal pupil reactivity, two indicators of structural and functional damage. C3 fragments and TCC increased, respectively, by 3.5 - and 4.0-fold in TBI compared to non-TBI tissue and significantly correlated with MBL, ficolin-2, ficolin-3, MASP-2 and MASP-3 levels in the homogenates. In conclusion, we show for the first time the direct presence of lectin pathway components in human cerebral contusions and their association with injury severity, suggesting a central role for the lectin pathway in the post-traumatic pathophysiology of human TBI.

Ultrasound-tagged near-infrared spectroscopy does not disclose absent cerebral circulation in brain-dead adults

Background: Near‐infrared spectroscopy, a non‐invasive technique for monitoring cerebral oxygenation, is widely used, but its accuracy is questioned because of the possibility of extra‐cranial contamination. Ultrasound‐tagged near‐infrared spectroscopy (UT‐NIRS) has been proposed as an improvement over previous methods. We investigated UT‐NIRS in healthy volunteers and in brain‐dead patients. Methods: We studied 20 healthy volunteers and 20 brain‐dead patients with two UT‐NIRS devices, CerOx™ and c‐FLOW™ (Ornim Medical, Kfar Saba, Israel), which measure cerebral flow index (CFI), a parameter related to changes in cerebral blood flow (CBF). Monitoring started after the patients had been declared brain dead for a median of 34 (range: 11–300) min. In 11 cases, we obtained further demonstration of absent CBF. Results: In healthy volunteers, CFI was markedly different in the two hemispheres in the same subject, with wide variability amongst subjects. In brain‐dead patients (median age: 64 yr old, 45% female; 20% traumatic brain injury, 40% subarachnoid haemorrhage, and 40% intracranial haemorrhage), the median (inter‐quartile range) CFI was 41 (36–47), significantly higher than in volunteers (33; 27–36). Conclusions: In brain‐dead patients, where CBF is absent, the UT‐NIRS findings can indicate an apparently perfused brain. This might reflect an insufficient separation of signals from extra‐cranial structures from a genuine appraisal of cerebral perfusion. For non‐invasive assessment of CBF‐related parameters, the near‐infrared spectroscopy still needs substantial improvement.