Cerebral ischemia entails rapid tissue damage in the affected brain area causing devastating neurological dysfunction. How each component of the neurovascular unit contributes or responds to the ischemic insult in the context of the human brain has not been solved yet. Thus, the analysis of the proteome is a straightforward approach to unraveling these cell proteotypes. In this study, post-mortem brain slices from ischemic stroke patients were obtained corresponding to infarcted (IC) and contralateral (CL) areas. By means of laser microdissection, neurons and blood brain barrier structures (BBB) were isolated and analyzed using label-free quantification. MS data are available via ProteomeXchange with identifier PXD003519. Ninety proteins were identified only in neurons, 260 proteins only in the BBB and 261 proteins in both cell types. Bioinformatics analyses revealed that repair processes, mainly related to synaptic plasticity, are outlined in microdissected neurons, with nonexclusive important functions found in the BBB. A total of 30 proteins showing p < 0.05 and fold-change> 2 between IC and CL areas were considered meaningful in this study: 13 in neurons, 14 in the BBB and 3 in both cell types. Twelve of these proteins were selected as candidates and analyzed by immunohistofluorescence in independent brains. The MS findings were completely verified for neuronal SAHH2 and SRSF1 whereas the presence in both cell types of GABT and EAA2 was only validated in neurons. In addition, SAHH2 showed its potential as a prognostic biomarker of neurological improvement when analyzed early in the plasma of ischemic stroke patients. Therefore, the quantitative proteomes of neurons and the BBB (or proteotypes) after human brain ischemia presented here contribute to increasing the knowledge regarding the molecular mechanisms of ischemic stroke pathology and highlight new proteins that might represent putative biomarkers of brain ischemia or therapeutic targets.

After stroke and other brain injuries, there is a high incidence of respiratory complications such as pneumonia or acute lung injury. The molecular mechanisms that drive the brain-lung interaction post-stroke have not yet been elucidated. We performed transient middle cerebral artery occlusion (MCAO) and sham surgery on C57BL/6J mice and collected bronchoalveolar lavage fluid (BALF), serum, brain, and lung homogenate samples 24 h after surgery. A 92 proteins-panel developed by Olink Proteomics^® was used to analyze the content in BALF and lung homogenates. MCAO animals had higher protein concentration levels in BALF than sham-controls, but these levels did not correlate with the infarct volume. No alteration in alveolar-capillary barrier permeability was observed. A total of 12 and 14 proteins were differentially expressed between the groups (FDR < 0.1) in BALF and lung tissue homogenates, respectively. Of those, HGF, TGF-α, and CCL2 were identified as the most relevant to this study. Their protein expression patterns were verified by ELISA. This study confirmed that post-stroke lung damage was not associated with increased lung permeability or cerebral ischemia severity. Furthermore, the dysregulation of HGF, TGF-α, and CCL2 in BALF and lung tissue after ischemia could play an important role in the molecular mechanisms underlying stroke-induced lung damage.

Cerebral amyloid angiopathy (CAA) is a major cause of lobar intracerebral hemorrhage (ICH) in elderly patients. Growing evidence suggests a potential role of aquaporin 4 (AQP4) in amyloid-beta-associated diseases, including CAA pathology. Our aim was to investigate the circulating levels of AQP4 in a cohort of patients who had suffered a lobar ICH with a clinical diagnosis of CAA. AQP4 levels were analyzed in the serum of 60 CAA-related ICH patients and 19 non-stroke subjects by enzyme-linked immunosorbent assay (ELISA). The CAA–ICH cohort was divided according to the time point of the functional outcome evaluation: mid-term (12 ± 18.6 months) and long-term (38.5 ± 32.9 months) after the last ICH. Although no differences were found in AQP4 serum levels between cases and controls, lower levels were found in CAA patients presenting specific hemorrhagic features such as ≥2 lobar ICHs and ≥5 lobar microbleeds detected by magnetic resonance imaging (MRI). In addition, CAA-related ICH patients who presented a long-term good functional outcome had higher circulating AQP4 levels than subjects with a poor outcome or controls. Our data suggest that AQP4 could potentially predict a long-term functional outcome and may play a protective role after a lobar ICH.

Traumatic brain injury (TBI) is a highly heterogeneous disease and achieving an accurate diagnosis remains a significant challenge. Biomarkers play a crucial role in minimizing the reliance on invasive techniques like computed tomography, which also have significant economic costs. Human samples were obtained from prospective cohort studies. Mice were subjected to an experimental model of traumatic brain injury. Biomarker levels, gene expression, and blood–brain barrier integrity were analyzed using ELISA, qRT-PCR, and Evans Blue assay; data were statistically evaluated using parametric or non-parametric tests as appropriate. This study focuses on evaluating the role of matricellular protein thrombospondin-1 (TSP-1) and the tight junction proteins occludin and ZO-1 as potential biomarkers of TBI. We showed that lower serum TSP-1 levels correlated with poor patient outcomes at 6 months compared to those patients with a good outcome. Additionally, the disruption of the blood–brain barrier (BBB) and subsequent release of tight junction proteins allowed us to identify occludin as a potential biomarker for prognosis in a cohort of TBI patients and as a diagnosis biomarker in a subgroup of patients with mild TBI, but its discriminative power as a diagnosis biomarker appears modest, as reflected by an AUC of 0.693. On the other hand, ZO-1 exhibited increased levels but limited diagnostic utility. These findings highlight the critical role of TSP-1 in maintaining BBB integrity and regulating the inflammatory response after a TBI, supported by the worsened condition observed in TSP-1-deficient animals. These results demonstrate the potential of TSP-1 and occludin as valuable biomarkers for secondary injury and disease progression in patients with mild to moderate/severe TBI.

Background: Traumatic brain injury (TBI) is a highly heterogeneous disease and achieving an accurate diagnosis remains a significant challenge. Biomarkers play a crucial role in minimizing the reliance on invasive techniques like computed tomography, which also have significant economic costs. Methods: Human samples were obtained from prospective cohort studies. Mice were subjected to an experimental model of traumatic brain injury. Biomarker levels, gene expression, and blood-brain barrier integrity were analyzed using ELISA, qRT-PCR, and Evans Blue assay; data were statistically evaluated using parametric or non-parametric tests as appropriate. Results: This work focuses on evaluating the role of matricellular protein thrombospondin-1 (TSP-1) and the tight junction proteins occludin and ZO-1 as potential biomarkers of TBI. We showed that lower serum TSP-1 levels correlated with poor patient outcome at 6 months compared to those patients with a good outcome. Additionally, the disruption of the blood-brain barrier (BBB) and subsequent release of tight junction proteins allowed us to identify occludin as a potential biomarker for prognosis in a cohort of TBI patients and as a diagnosis biomarker in a subgroup of patients with mild TBI. Conclusions: These findings highlight the critical role of TSP-1 in maintaining the BBB integrity and regulating the inflammatory response after TBI, supported by the worsened condition observed in TSP-1-deficient animals. These results demonstrate the potential of TSP-1 and occludin as valuable biomarkers for secondary injury and disease progression in patients with mild to moderate-severe TBI.