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Aims: Cerebral complications after cardiac arrest (CA) remain a major problem worldwide. The aim was to test the effects of sodium-ß-hydroxybutyrate (SBHB) infusion on brain injury in a clinically relevant swine model of CA., Results: CA was electrically induced in 20 adult swine. After 10 min, cardiopulmonary resuscitation was performed for 5 min. After return of spontaneous circulation (ROSC), the animals were randomly assigned to receive an infusion of balanced crystalloid (controls, n = 11) or SBHB (theoretical osmolarity 1189 mOsm/l, n = 8) for 12 h. Multimodal neurological and cardiovascular monitoring were implemented in all animals. Nineteen of the 20 animals achieved ROSC. Blood sodium concentrations, osmolarity and circulating KBs were higher in the treated animals than in the controls. SBHB infusion was associated with significantly lower plasma biomarkers of brain injury at 6 (glial fibrillary acid protein, GFAP and neuron specific enolase, NSE) and 12 h (neurofilament light chain, NFL, GFAP and NSE) compared to controls. The amplitude of the stereoelectroencephalograph (sEEG) increased in treated animals after ROSC compared to controls. Cerebral glucose uptake was lower in treated animals., Conclusions: In this experimental model, SBHB infusion after resuscitated CA was associated with reduced circulating markers of cerebral injury and increased sEEG amplitude., (© 2024. The Author(s).)

Traumatic brain injury (TBI) is an alteration of brain function caused by a sudden transmission of an external force to the head. The biomechanical impact induces acute and chronic metabolic changes that highly contribute to injury evolution and outcome. TBI heterogeneity calls for approaches allowing the mapping of regional molecular and metabolic changes underpinning disease progression, with mass spectrometry imaging (MSI) as an efficient tool to study the spatial distribution of small metabolites. In this study, we applied an innovative targeted atmospheric pressure-MALDI mass spectrometry imaging (AP-MALDI MSI) approach, starting from an extensive list of metabolites, representative of different metabolic pathways, individually validated on the tissue under analysis with original standards using 2,5-dihydroxybenzoic acid (DHB), to characterize the impact of TBI on regional changes to small metabolites in the brain. Brains from sham and TBI mice obtained 21 days post-injury were analyzed to examine the spatial metabolic profile of small metabolites belonging to different metabolic pathways. By a whole brain analysis, we identified four metabolites (alanine, lysine, histidine, and inosine) with higher abundance in TBI than sham mice. Within the TBI group, lysine, histidine, and inosine were higher in the hemisphere ipsilateral to the biomechanical impact vs. the contralateral one. Images showed a major involvement of the ipsilateral thalamus characterized by the increase of arginine, lysine, histidine, and inosine and a significant reduction of glutamic acid, and N-acetylaspartic acid compared to the contralateral thalamus. These findings indicate high-resolution imaging mass spectrometry as a powerful tool to identify region-specific changes after a TBI to understand the metabolic changes underlying brain injury evolution., (© 2024. The Author(s).)

The lectin pathway (LP) of complement mediates inflammatory processes linked to tissue damage and loss of function following traumatic brain injury (TBI). LP activation triggers a cascade of proteolytic events initiated by LP specific enzymes called MASPs (for Mannan-binding lectin Associated Serine Proteases). Elevated serum and brain levels of MASP-2, the effector enzyme of the LP, were previously reported to be associated with the severity of tissue injury and poor outcomes in patients with TBI. To evaluate the therapeutic potential of LP inhibition in TBI, we first conducted a pilot study testing the effect of an inhibitory MASP-2 antibody (α-MASP-2), administered systemically at 4 and 24 h post-TBI in a mouse model of controlled cortical impact (CCI). Treatment with α-MASP-2 reduced sensorimotor and cognitive deficits for up to 5 weeks post-TBI. As previous studies by others postulated a critical role of MASP-1 in LP activation, we conducted an additional study that also assessed treatment with an inhibitory MASP-1 antibody (α-MASP-1). A total of 78 mice were treated intraperitoneally with either α-MASP-2, or α-MASP-1, or an isotype control antibody 4 h and 24 h after TBI or sham injury. An amelioration of the cognitive deficits assessed by Barnes Maze, prespecified as the primary study endpoint, was exclusively observed in the α-MASP-2-treated group. The behavioral data were paralleled by a reduction of the lesion size when evaluated histologically and by reduced systemic LP activity. Our data suggest that inhibition of the LP effector enzyme MASP-2 is a promising treatment strategy to limit neurological deficits and tissue loss following TBI. Our work has translational value because a MASP-2 antibody has already completed multiple late-stage clinical trials in other indications and we used a clinically relevant treatment protocol testing the therapeutic mechanism of MASP-2 inhibition in TBI., (© 2024. The Author(s).)

Background: Traumatic brain injury (TBI) is a significant cause of death and disability, with no effective neuroprotective drugs currently available for its treatment. Mesenchymal stromal cell (MSC)-based therapy shows promise as MSCs release various soluble factors that can enhance the injury microenvironment through processes, such as immunomodulation, neuroprotection, and brain repair. Preclinical studies across different TBI models and severities have demonstrated that MSCs can improve functional and structural outcomes. Moreover, clinical evidence supports the safety of third-party donor bank-stored MSCs in adult subjects. Building on this preclinical and clinical data, we present the protocol for an academic, investigator-initiated, multicenter, double-blind, randomised, placebo-controlled, adaptive phase II dose-finding study aiming to evaluate the safety and efficacy of intravenous administration of allogeneic bone marrow-derived MSCs to severe TBI patients within 48 h of injury., Methods/design: The study will be conducted in two steps. Step 1 will enrol 42 patients, randomised in a 1:1:1 ratio to receive 80 million MSCs, 160 million MSCs or a placebo to establish safety and identify the most promising dose. Step 2 will enrol an additional 36 patients, randomised in a 1:1 ratio to receive the selected dose of MSCs or placebo. The activity of MSCs will be assessed by quantifying the plasmatic levels of neurofilament light (NfL) at 14 days as a biomarker of neuronal damage. It could be a significant breakthrough if the study demonstrates the safety and efficacy of MSC-based therapy for severe TBI patients. The results of this trial could inform the design of a phase III clinical trial aimed at establishing the efficacy of the first neurorestorative therapy for TBI., Discussion: Overall, the MATRIx trial is a critical step towards developing an effective treatment for TBI, which could significantly improve the lives of millions worldwide affected by this debilitating condition. Trial Registration EudraCT: 2022-000680-49., (© 2023. European Society of Intensive Care Medicine and Springer Nature Switzerland AG.)

Background: Traumatic brain injury (TBI) is a leading cause of death and long-term disability worldwide. In addition to primary brain damage, systemic immune alterations occur, with evidence for dysregulated immune responses in aggravating TBI outcome and complications. However, immune dysfunction following TBI has been only partially understood, especially in the elderly who represent a substantial proportion of TBI patients and worst outcome. Therefore, we aimed to conduct an in-depth immunological characterization of TBI patients, by evaluating both adaptive (T and B lymphocytes) and innate (NK and monocytes) immune cells of peripheral blood mononuclear cells (PBMC) collected acutely (< 48 h) after TBI in young (18-45 yo) and elderly (> 65 yo) patients, compared to age-matched controls, and also the levels of inflammatory biomarkers., Results: Our data show that young respond differently than elderly to TBI, highlighting the immune unfavourable status of elderly compared to young patients. While in young only CD4 T lymphocytes are activated by TBI, in elderly both CD4 and CD8 T cells are affected, and are induced to differentiate into subtypes with low cytotoxic activity, such as central memory CD4 T cells and memory precursor effector CD8 T cells. Moreover, TBI enhances the frequency of subsets that have not been previously investigated in TBI, namely the double negative CD27- IgD- and CD38-CD24- B lymphocytes, and CD56dim CD16- NK cells, both in young and elderly patients. TBI reduces the production of pro-inflammatory cytokines TNF-α and IL-6, and the expression of HLA-DM, HLA-DR, CD86/B7-2 in monocytes, suggesting a compromised ability to drive a pro-inflammatory response and to efficiently act as antigen presenting cells., Conclusions: We described the acute immunological response induced by TBI and its relation with injury severity, which could contribute to pathologic evolution and possibly outcome. The focus on age-related immunological differences could help design specific therapeutic interventions based on patients' characteristics., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Traumatic brain injury (TBI) is a major worldwide neurological disorder with no neuroprotective treatment available. Three-dimensional (3D) in vitro models of brain contusion serving as a screening platform for drug testing are lacking. Here we developed a new in vitro model of brain contusion on organotypic cortical brain slices and tested its responsiveness to mesenchymal stromal cell (MSC) derived secretome. A focal TBI was induced on organotypic slices by an electromagnetic impactor. Compared to control condition, a temporal increase in cell death was observed after TBI by propidium iodide incorporation and lactate dehydrogenase release assays up to 48 h post-injury. TBI induced gross neuronal loss in the lesion core, with disruption of neuronal arborizations measured by microtubule-associated protein-2 (MAP-2) immunostaining and associated with MAP-2 gene down-regulation. Neuronal damage was confirmed by increased levels of neurofilament light chain (NfL), microtubule associated protein (Tau) and ubiquitin C-terminal hydrolase L1 (UCH-L1) released into the culture medium 48 h after TBI. We detected glial activation with microglia cells acquiring an amoeboid shape with less ramified morphology in the contusion core. MSC-secretome treatment, delivered 1 h post-injury, reduced cell death in the contusion core, decreased NfL release in the culture media, promoted neuronal reorganization and improved microglia survival/activation. Our 3D in vitro model of brain contusion recapitulates key features of TBI pathology. We showed protective effects of MSC-secretome, suggesting the model stands as a tractable medium/high throughput, ethically viable, and pathomimetic biological asset for testing new cell-based therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pischiutta, Cavaleiro, Caruso, Tribuzio, Di Marzo, Moro, Kobeissy, Wang, Salgado and Zanier.)

Mild traumatic brain injury (mTBI) mostly causes transient symptoms, but repeated (r)mTBI can lead to neurodegenerative processes. Diagnostic tools to evaluate the presence of ongoing occult neuropathology are lacking. In a mouse model of rmTBI, we investigated MRI and plasma biomarkers of brain damage before chronic functional impairment arose. Anesthetized adult male and female C57BL/6J mice were subjected to rmTBI or a sham procedure. Sensorimotor deficits were evaluated up to 12 months post-injury in SNAP and Neuroscore tests. Cognitive function was assessed in the novel object recognition test at six and 12 months. Diffusion tensor imaging (DTI) and structural magnetic resonance imaging (MRI) were performed at six and 12 months to examine white matter and structural damage. Plasma levels of neurofilament light (NfL) were assessed longitudinally up to 12 months. Brain histopathology was performed at 12 months. Independent groups of mice were used to examine the effects of 2-, 7- and 14-days inter-injury intervals on acute plasma NfL levels and on hyperactivity. Twelve months after an acute transient impairment, sensorimotor functions declined again in rmTBI mice ( p  < 0.001 vs sham), but not earlier. Similarly, rmTBI mice showed memory impairment at 12 ( p  < 0.01 vs sham) but not at 6 months. White matter damage examined by DTI was evident in rmTBI mice at both six and 12 months ( p  < 0.001 vs sham). This was associated with callosal atrophy ( p  < 0.001 vs sham) evaluated by structural MRI. Plasma NfL at one week was elevated in rmTBI ( p  < 0.001 vs sham), and its level correlated with callosal atrophy at 12 months (Pearson r = 0.72, p  < 0.01). Histopathology showed thinning of the corpus callosum and marked astrogliosis in rmTBI mice. The NfL levels were higher in mice subjected to short (2 days) compared with longer (7 and 14 days) inter-injury intervals ( p  < 0.05), and this correlated with hyperactivity in mice (Pearson r = 0.50; p < 0.05). These findings show that rmTBI causes white matter pathology detectable by MRI before chronic functional impairment. Early quantification of plasma NfL correlates with the degree of white matter atrophy one year after rmTBI and can serve to monitor the brain's susceptibility to a second mTBI, supporting its potential clinical application to guide the return to practice in sport-related TBI.

Brain ischemia is a common acute injury resulting from impaired blood flow to the brain. Translation of effective drug candidates from experimental models to patients has systematically failed. The use of human induced pluripotent stem cells (iPSC) offers new opportunities to gain translational insights into diseases including brain ischemia. We used a human 3D self-assembling iPSC-derived model (human cortical organoids, hCO) to characterize the effects of ischemia caused by oxygen-glucose deprivation (OGD). hCO exposed to 2 h or 8 h of OGD had neuronal death and impaired neuronal network complexity, measured in whole-mounting microtubule-associated protein 2 (MAP-2) immunostaining. Neuronal vulnerability was reflected by a reduction in MAP-2 mRNA levels, and increased release of neurofilament light chain (NfL) in culture media, proportional to OGD severity. Glial fibrillary acidic protein (GFAP) gene or protein levels did not change in hCO, but their release in medium increased after prolonged OGD. In conclusion, this human 3D iPSC-based in vitro model of brain ischemic injury is characterized by marked neuronal injury reflected by the release of the translational biomarker NfL which is relevant for testing neuroprotective strategies.

Introduction: Prognosis after resuscitation from cardiac arrest (CA) remains poor, with high morbidity and mortality as a result of extensive cardiac and brain injury and lack of effective treatments. Hypertonic sodium lactate (HSL) may be beneficial after CA by buffering severe metabolic acidosis, increasing brain perfusion and cardiac performance, reducing cerebral swelling, and serving as an alternative energetic cellular substrate. The aim of this study was to test the effects of HSL infusion on brain and cardiac injury in an experimental model of CA., Methods: After a 10-min electrically induced CA followed by 5 min of cardiopulmonary resuscitation maneuvers, adult swine (n = 35) were randomly assigned to receive either balanced crystalloid (controls, n = 11) or HSL infusion started during cardiopulmonary resuscitation (CPR, Intra-arrest, n = 12) or after return of spontaneous circulation (Post-ROSC, n = 11) for the subsequent 12 h. In all animals, extensive multimodal neurological and cardiovascular monitoring was implemented. All animals were treated with targeted temperature management at 34 °C., Results: Thirty-four of the 35 (97.1%) animals achieved ROSC; one animal in the Intra-arrest group died before completing the observation period. Arterial pH, lactate and sodium concentrations, and plasma osmolarity were higher in HSL-treated animals than in controls (p < 0.001), whereas potassium concentrations were lower (p = 0.004). Intra-arrest and Post-ROSC HSL infusion improved hemodynamic status compared to controls, as shown by reduced vasopressor requirements to maintain a mean arterial pressure target > 65 mmHg (p = 0.005 for interaction; p = 0.01 for groups). Moreover, plasma troponin I and glial fibrillary acid protein (GFAP) concentrations were lower in HSL-treated groups at several time-points than in controls., Conclusions: In this experimental CA model, HSL infusion was associated with reduced vasopressor requirements and decreased plasma concentrations of measured biomarkers of cardiac and cerebral injury., (© 2023. The Author(s).)

The severity and long-term consequences of brain damage in traumatic brain injured (TBI) patients urgently calls for better neuroprotective/neuroreparative strategies for this devastating disorder. Mesenchymal stromal cells (MSCs) hold great promise and have been shown to confer neuroprotection in experimental TBI, mainly through paracrine mechanisms via secreted bioactive factors (i.e. secretome), which indicates significant potential for a cell-free neuroprotective approach. The secretome is composed of cytokines, chemokines, growth factors, proteins, lipids, nucleic acids, metabolites, and extracellular vesicles; it may offer advantages over MSCs in terms of delivery, safety, and variability of therapeutic response for brain injury. Immunomodulation by molecular factors secreted by MSCs is considered to be a key mechanism involved in their multi-potential therapeutic effects. Regulated neuroinflammation is required for healthy remodeling of central nervous system during development and adulthood. Moreover, immune cells and their secreted factors can also contribute to tissue repair and neurological recovery following acute brain injury. However, a chronic and maladaptive neuroinflammatory response can exacerbate TBI and contribute to progressive neurodegeneration and long-term neurological impairments. Here, we review the evidence for MSC-derived secretome as a therapy for TBI. Our framework incorporates a detailed analysis of in vitro and in vivo studies investigating the effects of the secretome on clinically relevant neurological and histopathological outcomes. We also describe the activation of immune cells after TBI and the immunomodulatory properties exerted by mediators released in the secretome. We then describe how ageing modifies central and systemic immune responses to TBI and discuss challenges and opportunities of developing secretome based neuroprotective therapies for elderly TBI populations. Finally, strategies aimed at modulating the secretome in order to boost its efficacy for TBI will also be discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Traumatic brain injury is increasingly common in older individuals. Older age is one of the strongest predictors for poor prognosis after brain trauma, a phenomenon driven by the presence of extra-cranial comorbidities as well as pre-existent pathologies associated with cognitive impairment and brain volume loss (such as cerebrovascular disease or age-related neurodegeneration). Furthermore, ageing is associated with a dysregulated immune response, which includes attenuated responses to infection and vaccination, and a failure to resolve inflammation leading to chronic inflammatory states. In traumatic brain injury, where the immune response is imperative for the clearance of cellular debris and survey of the injured milieu, an appropriate self-limiting response is vital to promote recovery. Currently, our understanding of age-related factors that contribute to the outcome is limited; but a more complete understanding is essential for the development of tailored therapeutic strategies to mitigate the consequences of traumatic brain injury. Here we show greater functional deficits, white matter abnormalities and worse long-term outcomes in aged compared with young C57BL/6J mice after either moderate or severe traumatic brain injury. These effects are associated with altered systemic, meningeal and brain tissue immune response. Importantly, the impaired acute systemic immune response in the mice was similar to the findings observed in our clinical cohort. Traumatic brain-injured patient cohort over 70 years of age showed lower monocyte and lymphocyte counts compared with those under 45 years. In mice, traumatic brain injury was associated with alterations in peripheral immune subsets, which differed in aged compared with adult mice. There was a significant increase in transcription of immune and inflammatory genes in the meninges post-traumatic brain injury, including monocyte/leucocyte-recruiting chemokines. Immune cells were recruited to the region of the dural injury, with a significantly higher number of CD11b + myeloid cells in aged compared with the adult mice. In brain tissue, when compared with the young adult mice, we observed a more pronounced and widespread reactive astrogliosis 1 month after trauma in aged mice, sustained by an early and persistent induction of proinflammatory astrocytic state. These findings provide important insights regarding age-related exacerbation of neurological damage after brain trauma., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Mesenchymal stromal cells (MSCs) are widely used in preclinical models of traumatic brain injury (TBI). Results are promising in terms of neurological improvement but are hampered by wide variability in treatment responses. We made a systematic review and meta-analysis: (1) to assess the quality of evidence for MSC treatment in TBI rodent models; (2) to determine the effect size of MSCs on sensorimotor function, cognitive function, and anatomical damage; (3) to identify MSC-related and protocol-related variables associated with greater efficacy; (4) to understand whether MSC manipulations boost therapeutic efficacy. The meta-analysis included 80 studies. After TBI, MSCs improved sensorimotor and cognitive deficits and reduced anatomical damage. Stratified meta-analysis on sensorimotor outcome showed similar efficacy for different MSC sources and for syngeneic or xenogenic transplants. Efficacy was greater when MSCs were delivered in the first-week post-injury, and when implanted directly into the lesion cavity. The greatest effect size was for cells embedded in matrices or for MSC-derivatives. MSC therapy is effective in preclinical TBI models, improving sensorimotor, cognitive, and anatomical outcomes, with large effect sizes. These findings support clinical studies in TBI., (© 2021. The Author(s).)

The multiplicity of systems affected in Alzheimer's disease (AD) brains calls for multi-target therapies. Although mesenchymal stem cells (MSC) are promising candidates, their clinical application is limited because of risks related to their direct implantation in the host. This could be overcome by exploiting their paracrine action. We herein demonstrate that in vivo systemic administration of secretome collected from MSC exposed in vitro to AD mouse brain homogenates (MSC-CS), fully replicates the cell-mediated neuroreparative effects in APP/PS1 AD mice. We found a complete but transient memory recovery by 7 days, which vanished by 14 days, after a single MSC-CS intravenous administration in 12-month or 22-24-month-old mice. Treatment significantly reduced plaque load, microglia activation, and expression of cytokines in astrocytes in younger, but not aged, mice at 7 days. To optimize efficacy, we established a sustained treatment protocol in aged mice through intranasal route. Once-weekly intranasal administration of MSC-CS induced persistent memory recovery, with dramatic reduction of plaques surrounded by a lower density of β-amyloid oligomers. Gliosis and the phagocytic marker CD68 were decreased. We found a higher neuronal density in cortex and hippocampus, associated with a reduction in hippocampal shrinkage and a longer lifespan indicating healthier conditions of MSC-CS-treated compared to vehicle-treated APP/PS1 mice. Our data prove that MSC-CS displays a great multi-level therapeutic potential, and lay the foundation for identifying the therapeutic secretome bioreactors leading to the development of an efficacious multi-reparative cocktail drug, towards abrogating the need for MSC implantation and risks related to their direct use.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. In the last 30 years several neuroprotective agents, attenuating the downstream molecular and cellular damaging events triggered by TBI, have been extensively studied. Even though many drugs have shown promising results in the pre-clinical stage, all have failed in large clinical trials. Mesenchymal stromal cells (MSCs) may offer a promising new therapeutic intervention, with preclinical data showing protection of the injured brain. We selected three of the critical aspects identified as possible causes of clinical failure: the window of opportunity for drug administration, the double-edged contribution of mechanisms to damage and recovery, and the oft-neglected role of reparative mechanisms. For each aspect, we briefly summarized the limitations of previous trials and the potential advantages of a newer approach using MSCs.

Traumatic brain injury is a risk factor for subsequent neurodegenerative disease, including chronic traumatic encephalopathy, a tauopathy mostly associated with repetitive concussion and blast, but not well recognized as a consequence of severe traumatic brain injury. Here we show that a single severe brain trauma is associated with the emergence of widespread hyperphosphorylated tau pathology in a proportion of humans surviving late after injury. In parallel experimental studies, in a model of severe traumatic brain injury in wild-type mice, we found progressive and widespread tau pathology, replicating the findings in humans. Brain homogenates from these mice, when inoculated into the hippocampus and overlying cerebral cortex of naïve mice, induced widespread tau pathology, synaptic loss, and persistent memory deficits. These data provide evidence that experimental brain trauma induces a self-propagating tau pathology, which can be transmitted between mice, and call for future studies aimed at investigating the potential transmissibility of trauma associated tau pathology in humans.

There is increasing recognition that traumatic brain injury (TBI) may initiate long-term neurodegenerative processes, particularly chronic traumatic encephalopathy. However, insight into the mechanisms transforming an initial biomechanical injury into a neurodegenerative process remain elusive, partly as a consequence of the paucity of informative pre-clinical models. This study shows the functional, whole brain imaging and neuropathological consequences at up to one year survival from single severe TBI by controlled cortical impact in mice. TBI mice displayed persistent sensorimotor and cognitive deficits. Longitudinal T2 weighted magnetic resonance imaging (MRI) showed progressive ipsilateral (il) cortical, hippocampal and striatal volume loss, with diffusion tensor imaging demonstrating decreased fractional anisotropy (FA) at up to one year in the il-corpus callosum (CC: -30%) and external capsule (EC: -21%). Parallel neuropathological studies indicated reduction in neuronal density, with evidence of microgliosis and astrogliosis in the il-cortex, with further evidence of microgliosis and astrogliosis in the il-thalamus. One year after TBI there was also a decrease in FA in the contralateral (cl) CC (-17%) and EC (-13%), corresponding to histopathological evidence of white matter loss (cl-CC: -68%; cl-EC: -30%) associated with ongoing microgliosis and astrogliosis. These findings indicate that a single severe TBI induces bilateral, long-term and progressive neuropathology at up to one year after injury. These observations support this model as a suitable platform for exploring the mechanistic link between acute brain injury and late and persistent neurodegeneration., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Spontaneous intracerebral haemorrhage (ICH) is a relatively common fatal disease, with an overall global incidence estimated at 24.6 per 100,000 person- years. Given the high degree of morbidity and mortality associated with ICH, therapies that may have neuroprotective effects are of increasing interest to clinicians. In this last context, cell therapies offer the promise of improving the disease course which cannot be addressed adequately by existing treatments., Objective: The aim of this review is to evaluate the protective effects and molecular mechanisms of mesenchymal stem cells (MSCs) on haemorrhagic brain following ICH. We also discuss possible emerging therapeutic approaches worth of further research., Methods and Results: The available literature on the therapeutic potential of MSCs in ICH animal models clearly demonstrated that MSCs enhance the functional recovery and reduce the volume of the infarct size exerting anti-inflammatory and angiogenic properties. However, the quality of the original articles investigating the efficacy of stem cell therapies in ICH animal models is still poor and the lack of ICH clinical trial does not permit to reach any relevant conclusions., Conclusion: Further studies have to be implemented in order to achieve standardized methods of MSCs isolation, characterization and administration to improve ICH treatments with MSCs or MSC-derived products., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Acute brain injury resulting from ischemic/hemorrhagic or traumatic damage is one of the leading causes of mortality and disability worldwide and is a significant burden to society. Neuroprotective options to counteract brain damage are very limited in stroke and traumatic brain injury (TBI). Given the multifaceted nature of acute brain injury and damage progression, several therapeutic targets may need to be addressed simultaneously to interfere with the evolution of the injury and improve the patient's outcome. Stem cells are ideal candidates since they act on various mechanisms of protection and repair, improving structural and functional outcomes after experimental stroke or TBI. Stem cells isolated from placenta offer advantages due to their early embryonic origin, ease of procurement, and ethical acceptance. We analyzed the evidence for the beneficial effects of placenta-derived stem cells in acute brain injury, with the focus on experimental studies of TBI and stroke, the engineering strategies pursued to foster cell potential, and characterization of the bioactive molecules secreted by placental cells, known as their secretome, as an alternative cell-free strategy. Results from the clinical application of placenta-derived stem cells for acute brain injury and ongoing clinical trials are summarily discussed.

Transplantation of human bone marrow mesenchymal stromal cells (hBM-MSC) promotes functional recovery after stroke in animal models, but the mechanisms underlying these effects remain incompletely understood. We tested the efficacy of Good Manufacturing Practices (GMP) compliant hBM-MSC, injected intravenously 3.5 hours after injury in mice subjected to transient middle cerebral artery occlusion (tMCAo). We addressed whether hBM-MSC are efficacious and if this efficacy is associated with cortical circuit reorganization using neuroanatomical analysis of GABAergic neurons (parvalbumin; PV-positive cells) and perineuronal nets (PNN), a specialized extracellular matrix structure which acts as an inhibitor of neural plasticity. tMCAo mice receiving hBM-MSC, showed early and lasting improvement of sensorimotor and cognitive functions compared to control tMCAo mice. Furthermore, 5 weeks post-tMCAo, hBM-MSC induced a significant rescue of ipsilateral cortical neurons; an increased proportion of PV-positive neurons in the perilesional cortex, suggesting GABAergic interneurons preservation; and a lower percentage of PV-positive cells surrounded by PNN, indicating an enhanced plastic potential of the perilesional cortex. These results show that hBM-MSC improve functional recovery and stimulate neuroprotection after stroke. Moreover, the downregulation of "plasticity brakes" such as PNN suggests that hBM-MSC treatment stimulates plasticity and formation of new connections in the perilesional cortex.

Traumatic brain injury (TBI) constitutes a major cause of death and long-term disability. At present, we lack methods to non-invasively track tissue biochemistry and hence select appropriate interventions for patients. We hypothesized that detailed label-free vibrational chemical analysis of focal TBI could provide such information. We assessed the early spatial and temporal changes in tissue biochemistry that are associated with brain injury in mice. Numerous differences were observed in the spectra of the contusion core and pericontusional tissue between 2 and 7 days. For example, a strong signal from haem was seen in the contusion core at 2 days due to haemorrhage, which subsequently resolved. More importantly, elevated cholesterol levels were demonstrated by 7 days, which may be a marker of important cell repair processes. Principal component analysis revealed an early 'acute' component dominated by haemorrhage and a delayed component reflecting changes in protein and lipid composition. Notably we demonstrated changes in Raman signature with time even in the contralateral hemisphere when compared to sham control mice. Raman spectroscopy therefore shows promise as a probe that is sensitive to important pathobiological processes in TBI and could be applied in future both in the experimental setting, as well as in the clinic.

Objectives: To define the features of human amniotic mesenchymal stromal cell secretome and its protective properties in experimental models of acute brain injury., Design: Prospective experimental study., Setting: Laboratory research., Subjects: C57Bl/6 mice., Interventions: Mice subjected to sham or traumatic brain injury by controlled cortical impact received human amniotic mesenchymal stromal cells or phosphate-buffered saline infused intracerebroventricularly or intravenously 24 hours after injury. Organotypic cortical brain slices exposed to ischemic injury by oxygen-glucose deprivation were treated with human amniotic mesenchymal stromal cells or with their secretome (conditioned medium) in a transwell system., Measurements and Main Results: Traumatic brain injured mice receiving human amniotic mesenchymal stromal cells intravenously or intracerebroventricularly showed early and lasting functional and anatomical brain protection. cortical slices injured by oxigen-glucose deprivation and treated with human amniotic mesenchymal stromal cells or conditioned medium showed comparable protective effects (neuronal rescue, promotion of M2 microglia polarization, induction of trophic factors) indicating that the exposure of human amniotic mesenchymal stromal cells to the injured tissue is not necessary for the release of bioactive factors. Using sequential size-exclusion and gel-filtration chromatography, we identified a conditioned medium subfraction, which specifically displays these highly protective properties and we found that this fraction was rich in bioactive molecules with molecular weight smaller than 700 Da. Quantitative RNA analysis and mass spectrometry-based peptidomics showed that the active factors are not proteins or RNAs. The metabolomic profiling of six metabolic classes identified a list of molecules whose abundance was selectively elevated in the active conditioned medium fraction., Conclusions: Human amniotic mesenchymal stromal cell-secreted factors protect the brain after acute injury. Importantly, a fraction rich in metabolites, and containing neither proteic nor ribonucleic molecules was protective. This study indicates the profiling of protective factors that could be useful in cell-free therapeutic approaches for acute brain injury.

In the cell therapy scenario, efficient tracing of transplanted cells is essential for investigating cell migration and interactions with host tissues. This is fundamental to provide mechanistic insights which altogether allow for the understanding of the translational potential of placental cell therapy in the clinical setting. Mesenchymal stem/stromal cells (MSC) from human placenta are increasingly being investigated for their potential in treating patients with a variety of diseases. In this study, we investigated the feasibility of using poly (methyl methacrylate) nanoparticles (PMMA-NPs) to trace placental MSC, namely those from the amniotic membrane (hAMSC) and early chorionic villi (hCV-MSC). We report that PMMP-NPs are efficiently internalized and retained in both populations, and do not alter cell morphofunctional parameters. We observed that PMMP-NP incorporation does not alter in vitro immune modulatory capability of placental MSC, a characteristic central to their reparative/therapeutic effects in vitro. We also show that in vitro, PMMP-NP uptake is not affected by hypoxia. Interestingly, after in vivo brain ischaemia and reperfusion injury achieved by transient middle cerebral artery occlusion (tMCAo) in mice, iv hAMSC treatment resulted in significant improvement in cognitive function compared to PBS-treated tMCAo mice. Our study provides evidence that tracing placental MSC with PMMP-NPs does not alter their in vitro and in vivo functions. These observations are grounds for the use of PMMP-NPs as tools to investigate the therapeutic mechanisms of hAMSC and hCV-MSC in preclinical models of inflammatory-driven diseases., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

An impaired ability to regulate microglia activation by fractalkine (CX3CL1) leads to microglia chronic sub-activation. How this condition affects outcome after acute brain injury is still debated, with studies showing contrasting results depending on the timing and the brain pathology. Here, we investigated the early and delayed consequences of fractalkine receptor (CX3CR1) deletion on neurological outcome and on the phenotypical features of the myeloid cells present in the lesions of mice with traumatic brain injury (TBI). Wild type (WT) and CX3CR1(-/-) C57Bl/6 mice were subjected to sham or controlled cortical impact brain injury. Outcome was assessed at 4 days and 5 weeks after TBI by neuroscore, neuronal count, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Compared with WT mice, CX3CR1(-/-) TBI mice showed a significant reduction of sensorimotor deficits and lower cellular damage in the injured cortex 4 days post-TBI. Conversely, at 5 weeks, they showed a worsening of sensorimotor deficits and pericontusional cell death. Microglia (M) and macrophage (μ) activation and polarization were assessed by quantitative immunohistochemistry for CD11b, CD68, Ym1, and inducible nitric oxide synthase (iNOS)-markers of M/μ activation, phagocytosis, M2, and M1 phenotypes, respectively. Morphological analysis revealed a decreased area and perimeter of CD11b(+) cells in CX3CR1(-/-) mice at 4 days post-TBI, whereas, at 5 weeks, both parameters were significantly higher, compared with WT mice. At 4 days, CX3CR1(-/-) mice showed significantly decreased CD68 and iNOS immunoreactivity, while at 5 weeks post-injury, they showed a selective increase of iNOS. Gene expression on CD11b(+) sorted cells revealed an increase of interleukin 10 and insulin-like growth factor 1 (IGF1) at 1 day and a decrease of IGF1 4 days and 5 weeks post-TBI in CX3CR1(-/-), compared with WT mice. These data show an early protection followed by a chronic exacerbation of TBI outcome in the absence of CX3CR1. Thus, longitudinal effects of myeloid cell manipulation at different stages of pathology should be investigated to understand how and when their modulation may offer therapeutic chances.

Many efforts have been performed in order to understand the role of recruited macrophages in the progression of spinal cord injury (SCI). Different studies revealed a pleiotropic effect played by these cells associated to distinct phenotypes (M1 and M2), showing a predictable spatial and temporal distribution in the injured site after SCI. Differently, the role of activated microglia in injury progression has been poorly investigated, mainly because of the challenges to target and selectively modulate them in situ. A delivery nanovector tool (poly-ε-caprolactone-based nanoparticles) able to selectively treat/target microglia has been developed and used here to clarify the temporal and spatial involvement of the pro-inflammatory response associated to microglial cells in SCI. We show that a treatment with nanoparticles loaded with minocycline, the latter a well-known anti-inflammatory drug, when administered acutely in a SCI mouse model is able to efficiently modulate the resident microglial cells reducing the pro-inflammatory response, maintaining a pro-regenerative milieu and ameliorating the behavioral outcome up to 63 days post injury. Furthermore, by using this selective delivery tool we demonstrate a mechanistic link between early microglia activation and M1 macrophages recruitment to the injured site via CCL2 chemokine, revealing a detrimental contribution of pro-inflammatory macrophages to injury progression after SCI., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The study of microglia and macrophage (M/M) morphology represents a key tool to understand the functional activation state and the pattern of distribution of these cells in acute brain injury. The identification of reliable quantitative morphological parameters is urgently needed to understand these cell roles in brain injury and to explore strategies aimed at therapeutically manipulating the inflammatory response., Methods: We used three different clinically relevant murine models of focal injury, namely, controlled cortical impact brain injury (traumatic brain injury (TBI)) and transient and permanent occlusion of middle cerebral artery (tMCAo and pMCAo, respectively). Twenty-four hours after injury, M/M cells were labeled by CD11b, and ×40 photomicrographs were acquired by unbiased sampling of the lesion core using a motorized stage microscope. Images were processed with Fiji software to obtain shape descriptors., Results: We validated several parameters, including area, perimeter, Feret's diameter (caliper), circularity, aspect ratio, and solidity, providing quantitative information on M/M morphology over wide tissue portions. We showed that the shape descriptors that best represent M/M ramification/elongation are area and perimeter, while circularity and solidity provide information on the ameboid shape. We also provide evidence of the involvement of different populations in local inflammatory events, with macrophages replacing microglia into the lesion core when reperfusion does not occur. Analysis of CD45(high)+ cell morphology, whose shape does not change, did not yield any difference, thus confirming the reliability of the approach., Conclusions: We have defined specific morphological features that M/M acquire in response to different acute insults by applying a sensitive and readily applicable approach to cell morphological analysis in the brain tissue. Potential application of this method can be extended to all cell types able to change shape following activation, e.g., astrocytes, or to different disease states, including chronic pathologies.

Cells of myeloid origin, such as microglia and macrophages, act at the crossroads of several inflammatory mechanisms during pathophysiology. Besides pro-inflammatory activity (M1 polarization), myeloid cells acquire protective functions (M2) and participate in the neuroprotective innate mechanisms after brain injury. Experimental research is making considerable efforts to understand the rules that regulate the balance between toxic and protective brain innate immunity. Environmental changes affect microglia/macrophage functions. Hypoxia can affect myeloid cell distribution, activity, and phenotype. With their intrinsic differences, microglia and macrophages respond differently to hypoxia, the former depending on ATP to activate and the latter switching to anaerobic metabolism and adapting to hypoxia. Myeloid cell functions include homeostasis control, damage-sensing activity, chemotaxis, and phagocytosis, all distinctive features of these cells. Specific markers and morphologies enable to recognize each functional state. To ensure homeostasis and activate when needed, microglia/macrophage physiology is finely tuned. Microglia are controlled by several neuron-derived components, including contact-dependent inhibitory signals and soluble molecules. Changes in this control can cause chronic activation or priming with specific functional consequences. Strategies, such as stem cell treatment, may enhance microglia protective polarization. This review presents data from the literature that has greatly advanced our understanding of myeloid cell action in brain injury. We discuss the selective responses of microglia and macrophages to hypoxia after stroke and review relevant markers with the aim of defining the different subpopulations of myeloid cells that are recruited to the injured site. We also cover the functional consequences of chronically active microglia and review pivotal works on microglia regulation that offer new therapeutic possibilities for acute brain injury.

Microglia/macrophages (M) are major contributors to postinjury inflammation, but they may also promote brain repair in response to specific environmental signals that drive classic (M1) or alternative (M2) polarization. We investigated the activation and functional changes of M in mice with traumatic brain injuries and receiving intracerebroventricular human bone marrow mesenchymal stromal cells (MSCs) or saline infusion. MSCs upregulated Ym1 and Arginase-1 mRNA (p < 0.001), two M2 markers of protective M polarization, at 3 and 7 d postinjury, and increased the number of Ym1(+) cells at 7 d postinjury (p < 0.05). MSCs reduced the presence of the lysosomal activity marker CD68 on the membrane surface of CD11b-positive M (p < 0.05), indicating reduced phagocytosis. MSC-mediated induction of the M2 phenotype in M was associated with early and persistent recovery of neurological functions evaluated up to 35 days postinjury (p < 0.01) and reparative changes of the lesioned microenvironment. In vitro, MSCs directly counteracted the proinflammatory response of primary murine microglia stimulated by tumor necrosis factor-α + interleukin 17 or by tumor necrosis factor-α + interferon-γ and induced M2 proregenerative traits, as indicated by the downregulation of inducible nitric oxide synthase and upregulation of Ym1 and CD206 mRNA (p < 0.01). In conclusion, we found evidence that MSCs can drive the M transcriptional environment and induce the acquisition of an early, persistent M2-beneficial phenotype both in vivo and in vitro. Increased Ym1 expression together with reduced in vivo phagocytosis suggests M selection by MSCs towards the M2a subpopulation, which is involved in growth stimulation and tissue repair.

The need for immunosuppression after allo/xenogenic mesenchymal stromal cell (MSC) transplantation is debated. This study compared the long-term effects of human (h) bone marrow MSC transplant in immunocompetent or immunosuppressed traumatic brain injured (TBI) mice. C57Bl/6 male mice were subjected to TBI or sham surgery followed 24 h later by an intracerebroventricular infusion of phosphate buffer saline (PBS, control) or hMSC (150,000/5 μl). Immunocompetent and cyclosporin A immunosuppressed (CsA) mice were analyzed for gene expression at 72 h, functional deficits and histological analysis at five weeks. Gene expression analysis showed the effectiveness of immunosuppression (INFγ reduction in CsA treated groups), with no evidence of early rejection (no changes of MHCII and CD86 in all TBI groups) and selective induction of T-reg (increase of Foxp3) only in the TBI hMSC group. Five weeks after TBI, hMSC had comparable efficacy, with functional recovery (on both sensorimotor and cognitive deficits) and structural protection (contusion volume, vessel rescue effect, gliotic scar reduction, induction of neurogenesis) in immunosuppressed and immunocompetent mice. Therefore, long-term hMSC efficacy in TBI is not dependent on immunosuppressive treatment. These findings could have important clinical implication since immunosuppression in acute TBI patients may increase their risk of infection and not be tolerated., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aims: To identify long-term sensorimotor and cognitive deficits and to evaluate structural alterations in brain ischemic mice., Methods: C57Bl/6J male mice were subjected to 30 min transient middle cerebral artery occlusion (tMCAo) or sham surgery. Sensorimotor deficits, exploratory behavior, and cognitive functions were evaluated up to 6 months. Cortical and subcortical damage were analyzed by MRI multiparameter analysis and histopathology., Results: tMCAo mice showed significant sensorimotor deficits in the rotarod, negative geotaxis, neuroscore, and beam walk tests. They also showed impairment in exploratory behavior in the open field test and in spatial learning in the Morris water maze. T2-weighted MRI revealed a volume reduction in injured brain areas at 12 and 24 weeks postinjury. Brain atrophy was shown by MRI and conventional postmortem analysis. Diffusion tensor imaging on the external capsule showed increased values of axial and radial diffusivity. Fiber tracking revealed a reduction in the number and length of ipsilateral fibers., Conclusions: tMCAo in mice induces sensorimotor and cognitive impairments detectable at least up to 6 months postinjury, associated with brain atrophy, and axonal and myelin damage of the external capsule. These behavioral tests and anatomical investigations may represent important tools in translational studies in cerebral ischemia., (© 2013 John Wiley & Sons Ltd.)