aSAH is a multifaceted and complex hemorrhagic cerebrovascular disease that carries high morbidity and mortality. Though we have been digging into this disorder for decades, treatment options remain limited. DCI occurs 4–10 days after hemorrhage and is the most predominant complication, severely affecting the patient’s prognosis. Recently, our understanding of the pathophysiology of DCI has undergone a paradigm shift away from vasospasm and toward a multifactorial process. Considering the complexity of the molecular pathological mechanism of DCI after aSAH, simultaneous analysis of different types of protein biomarkers is needed to describe it more precisely. This study is the first multi-targeted olink proteomic analysis of CSF from patients 5–7 d post-aSAH, and the results indicate the presence of multiple abnormal levels of proteins associated with neurology and inflammation in the CSF of aSAH patients, providing new evidence for the molecular pathological mechanisms and treatment of DCI after aSAH.

Based on cluster analysis and diverse functional enrichment analyses, we found that, compared with the control group, 18 of the differential proteins in the neurology panel were up-regulated in the CSF of the aSAH group, mainly related to phagosome, apoptosis, microRNAs in cancer pathway, etc., and 5 were down-regulated, primarily associated with neurotrophin signaling pathway and so on. Meantime, in the inflammation panel, all 31 differential proteins were up-regulated, predominantly involving the chemokine signaling pathway, viral protein interaction with cytokine and cytokine receptor, and toll-like receptor signaling pathway, etc. These differentially expressed proteins and pertinent pathways may be involved in diversified pathophysiological processes, which in turn mediate the development of DCI after aSAH. Next, we will elaborate on the specific context of the identified neuro- and inflammation-related proteins and their potential mechanisms in the pathological progression of DCI after aSAH, respectively.

Siglecs (sialic acid-binding immunoglobulin-type lectins) are a type I transmembrane receptor that typically binds to sialic acid. Siglecs are predominantly expressed in immune cells and generate activating or inhibitory signals [17, 18]. Recently, they have also been shown to be expressed on the surface of nervous system cells and play pivotal roles in neuroinflammation [19]. The human siglecs family consists of 15 members, which are basically divided into two groups: evolutionarily conserved siglecs and rapidly evolving siglecs [17]. Our results reveal remarkably elevated levels of Siglec-1 and Siglec-9 proteins in the CSF of patients with aSAH. Specifically, siglec-1 (sialoadherin or CD169) belongs to the conserved siglecs and is essentially a macrophage-restricted glycoprotein with a molecular weight of 200 kDa. Siglec-1 is the largest member of the siglecs family, with 16 C2-set domains, one V-set domain, a transmembrane domain, and a cytoplasmic tail [17, 20]. It has been reported that injury to the CNS, which destroys the blood-brain barrier, induces siglec-1 expression on a portion of macrophages and microglia within the parenchyma. The expression of siglec-1 matches the temporal and spatial distribution of the plasma extravasation into the brain parenchyma [21]. The latest study discovered that siglec-1 works synergistically with other macrophage receptors to promote phagocytosis [20]. Besides, CD169+ macrophages may control the inflammatory response through promoting interleukin-10 (IL-10) production [22]. However, the role and mechanism of Siglec-1 in aSAH have been rarely reported.

Siglec-9 is an evolutionary siglecs with only two C2-set domains, one V-set domain, one transmembrane domain, and a cytoplasmic tail. In humans, siglec-9 is functionally equivalent to siglec-E [17]. Of note, siglec-9 is uniquely expressed by human neutrophils and monocytes, as well as a minor population of natural killer cells [23]. The research found that siglec-E may be a crucial negative regulator of neutrophil recruitment and activation at the site of pneumonia [24, 25]. A plethora of studies have confirmed that neutrophils and the neutrophil extracellular traps (NETs) released by their activation are closely related to vascular injury and microthrombosis [26] and can mediate the occurrence of DCI after aSAH, while targeting neutrophils and NETs may ameliorate the DCI and the prognosis of patients with aSAH [27]. Therefore, we speculate that upregulating neutrophil siglec-9 may reverse the progression of DCI after aSAH by reducing the formation of NETs. Additionally, a recent study found that siglec-E is also expressed in microglia, and confirmed through in vivo and in vitro experiments that ablation of siglec-E can contribute to the activation of microglia and increase brain inflammation and ischemic injury [19], hinting that endogenously induced siglec-E exerts a key anti-inflammatory and neuroprotective role after ischemic stroke. Collectively, the elevated levels of siglec-1 and siglec-9 in CSF of aSAH patients may be the internal mechanism of cerebral inflammation regression and self-repair. Early activation of them may be an effective strategy to salvage the inflammatory microenvironment in the brain post-aSAH, prevent DCI, and improve the prognosis of patients.

Macrophage scavenger receptor 1 (MSR1), also known as CD204 or SR-A, is a homo-trimeric transmembrane glycoprotein consisting of six distinct domains [28]. It is mainly expressed in brain microglia and macrophages of other tissues and is responsible for inflammation regulation in various pathophysiological processes [29, 30]. Studies have confirmed that MSR1 has been shown to promote post-ischemic damage-associated molecular patterns (DAMPs, such as HMGB1 and Prxs) clearance, leading to the resolution of neuroinflammation and attenuation of ischemic brain injury in various animal models [31, 32]. Contrarily, in a spinal cord injury model, Kong et al. reported that macrophage MSR1 promotes phagocytosis of myelin debris and the formation of foamy macrophage, contributing to the pro-inflammatory polarization of macrophages and neuronal apoptosis, and exacerbating spinal cord injury [33]. The above contradictory results may be related to the distinct microenvironment and activation status of MSR1+ macrophages. Besides, in a rat model of subarachnoid hemorrhage, Tian et al. first covered that Msr1 expression was elevated in injured brain tissues, and knockdown of Msr1 could exacerbate neuroinflammation by promoting activation of the PI3K-Akt/NF-kb pathway and production of the inflammatory factors [34], dropping us a hint that Msr1 may conduce to the resolution of neuroinflammation after aSAH. Additionally, a notable increase of Msr1 level in the CSF of aSAH patients was discovered in the present study, whereas whether Msr1 reverses the progression of DCI after aSAH by promoting dissipation of neuroinflammation is unclear and deserves to be explored in depth.

Cathepsins are a group of proteases predominantly discovered in the endosomal-lysosomal system of mammalian tissue cells, and their main function is protein degradation in lysosomes [35]. There are 15 human cathepsins, including 11 cysteine proteases, 2 serine proteases and 2 aspartic proteases. Studies have confirmed that cathepsins not only support ongoing inflammatory and immune responses, and mediate neuropathological processes through their enzymatic activities, but are also involved in the maintenance of brain homeostasis [36]. Our results showed that the levels of cathepsin C (CTSC) and cathepsin S (CTSS) were both markedly elevated in the CSF of aSAH patients. Specifically, cathepsin C, also known as dipeptidyl peptidase I, is a cysteine exopeptidase that is expressed in immune and inflammatory cells (e.g. microglia, neutrophils, and cytotoxic T lymphocytes, etc.) [36, 37]. Plenty of in vivo and ex vivo experiments have confirmed that CTSC exacerbates neuroinflammation and mediates neurological injury as well as neurological dysfunction by contributing to the neurotoxic polarization of microglia [38,39,40], whereas the targeted inhibition of CTSC may be an effective strategy to control inflammation. However, the precise role of CTSC in aSAH has rarely been explored. Recent studies have found that CTSC regulates neutrophil infiltration and the formation of NETs [41], and NETs were reported to progressively increase over time, reaching a peak at day 7, in the CSF from patients with aSAH [42]. Taken together, we hypothesize that the production of CTSC in microglia and neutrophils may be responsible for the sustained inflammatory cycle accompanying pathogenesis in the brain. Cathepsin S, a member of cysteine cathepsins, is preferentially expressed in cells of mononuclear phagocytic origin, including macrophages, microglia and dendritic cells [36]. Recently, Xie et al. revealed by peripheral blood monocyte sequencing that monocytes with high expression of CTSS aggravated cerebral ischemia-reperfusion injury, whereas CTSS knockdown significantly ameliorated BBB disruption, vascular leakage, and reduced cerebral infarct area and neurological function scores [43]. Moreover, in a mouse model of traumatic brain injury (TBI), Xu et al. discovered that inhibition of CTSS markedly decreased the level of TBI-induced inflammatory factors in brain tissue and attenuated cerebral edema [44]. Of interest, although it has been reported that Cathepsin S is expressed in cerebral aneurysms and promotes the progression of cerebral aneurysms [45], its role and mechanism in secondary brain injury after aSAH are not clear. In summary, based on the aforementioned destructive role of CTSC and CTSS in multiple neurological disorders, we conjecture that these biomarkers are promising for the exploration of mechanisms of secondary pathological injury and therapeutic strategies post-aSAH and deserve further validation.

Interleukin-6 (IL-6) is a pleiotropic cytokine with roles in inflammation, immunity, neurovascular regeneration, and metabolism [46,47,48]. IL-6 is a small polypeptide (molecular weight of 19–28 kDa), comprised of four alpha helices and produced by lymphocytes, macrophages, including microglia, as well as fibroblasts, vascular endothelial cells, mast cells, and dendritic cells [49]. In most situations, it is considered to be a pro-inflammatory culprit affecting the pathogenesis of multiple CNS diseases [50, 51]. Current data show a more than 2,000-fold burst of increased IL-6 levels in CSF after SAH compared to controls. A growing body of evidence demonstrated that interleukin 6 in CSF is a biomarker for cerebral vasospasm, delayed cerebral ischemia (DCI) and related infarctions after aSAH [52,53,54], and designing effective anti-IL-6 strategies may be a new hope to salvage the above complications and ameliorate the prognosis of patients with aSAH.

Chemokines (or chemotactic cytokines) are a large family of small, secreted proteins (molecular weight of 8–10 kDa) that signal through cell surface G protein-coupled heptahelical chemokine receptors. Since their primary function is to mediate the migration of cells, especially leukocytes, they play central roles in all protective or destructive immune and inflammatory responses. The chemokine system comprises approximately 50 chemokine ligands and 20 chemokine receptors in humans. Based on the number and spacing of chemokine N-terminal cysteines, they are classified into four distinct subfamilies: CXCL, CCL, XCL, and CX3CL [55,56,57]. Several pieces of evidence revealed that chemokine signaling in the CNS exerts critical homeostatic and neuroprotective roles, and is expressed in neurons, glia and endothelial cells [57,58,59]. However, in some neuropathological situations such as ischemic stroke, chemokines also display significant neurotoxic or neurodestructive effects [60, 61]. Data from the current study indicate that chemokines (including CXCL1, CXCL6, CXCL8 (or IL-8), CXCL9, CXCL10, CXCL11, CCL2 (MCP-1), CCL3, CCL4, CCL8 (or MCP-2), CCL13 (or MCP-4), CCL19 and CCL23) are increased to varying degrees in the CSF of patients with aSAH compared to controls. Several of these chemokines have been reported to correlate with prognosis and other clinical parameters in patients with aSAH, while others have not been previously studied in the context of aSAH.

CXCL10, also known as interferon γ-induced protein 10 kDa (IP-10), stimulates the migration of monocytes and T cells to inflammatory tissues and exerts its biological effects by binding to CXCR3 [62]. In the present study, CXCL10 showed an intense activation, with a 37-fold increase in CSF in aSAH patients compared to controls. Consistent with our findings, Niwa et al. showed that IP-10 levels in the CSF of aSAH patients peaked on day 5, and that the dynamic alterations may be closely associated with the development of delayed ischemic neurological deficits after SAH [63]. Additionally, Spantler et al. recently revealed that serum IP-10 levels present marked associations with poor functional outcomes but not DCI after aSAH [64].

MCP-1(or CCL2), MCP-2 (CCL8), and MCP-4 (or CCL13) monocyte chemoattractant proteins [65], all of which were markedly elevated in the current study, hint that monocytes may play crucial roles in the intrathecal inflammatory response after aSAH. Mohme et al. once clarified that monocyte accumulation and activation in the CSF secondary to the early peak of CCL2 (day 3 after aSAH) correlated with DCI but also poor functional outcome [66]. Similar results were also reported by Niwa et al. [63]. and pointed to that elevated levels of IL-6 may induce the expression of MCP-1 in the CSF after SAH, followed by increases in the expression of IP-10 and MIG (namely CXCL-9, mainly responsible for T cells recruitment [66]). In conformity with our findings, higher serum levels of CCL2 were described to correlate with worse clinical outcomes in studies by Kim et al. in 2008 [67] and Ahn et al. in 2019 [68]. Notably, enhanced CSF levels of MCP-2 and MCP-4 on day 10 after aSAH also were connected with unfavorable outcomes (based on the Glasgow Outcome Scale) [12].

CXCL8 (or IL-8) and CXCL1 are principally responsible for chemotaxis of neutrophils [65, 66]. They all showed strong activation in CSF of aSAH patients in the present study. In line with our results, Mohme et al. reported that elevated concentrations of IL-8 and CXCL1 in the CSF were tightly relevant to the occurrence of DCI after aSAH [66]. Besides, Vlachogiannis et al. recently reported that enhanced CSF CXCL8 levels on day 10 after aSAH were associated with poor clinical outcomes at 1 year after SAH [12]. Although they also indicated persistently elevated levels of CXCL-1 in the CSF, it did not appear to be significantly correlated with the occurrence of DCI and clinical prognosis of patients with aSAH.

Furthermore, it is of interest that our data show that several tumor necrosis factor superfamily (TNFSF) members (known as potential drug targets in ischaemic vascular disease [69]) have elevated levels in CSF after aSAH, such as the ligands TNF beta (Tumor necrosis factor ligand superfamily member 1, TNFSF10) and TRAIL (Tumor necrosis factor ligand superfamily member 10, TNFSF10), receptors TNFRSF9 (Tumor necrosis factor receptor superfamily member 9), TNFRSF11B (Tumor necrosis factor receptor superfamily member 11B, also known as Osteoprotegerin, OPG) and CD40 (Tumor necrosis factor receptor superfamily member 5). Chen et al. once reported that higher plasma soluble CD40 ligand but not receptor levels correlate with clinical severity and may be a good prognostic biomarker for aSAH [70]. Moreover, a recent study also found that the above-mentioned members of the TNFSF have distinct expression patterns in the CSF after aSAH [14], but their relationship with the development of DCI and functional prognosis after aSAH has not yet been confirmed and deserves to be further explored in depth.