In this study we present a concise data set of the sequential production of multiple biomarkers of inflammation in the CSF of patients during the first 10 days post SAH. To our knowledge, this is the largest data set to describe the inflammatory profile of SAH ever reported with 92 inflammatory biomarkers included, many of which never studied in SAH patients previously. This is also the first report of Proximity Extension Assay (PEA) technology being used for measuring biomarker expression levels in the context of SAH. Similar studies were recently published in patients with traumatic brain injury (TBI) and trigeminal neuralgia, as well as numerous other non-neurosurgical conditions (such as neuropathic pain, cardiovascular diseases, gastric cancer, etc.).[29–31]
The analyses in the present study were performed in CSF compartment alone (not plasma or cerebral interstitial fluid) as this seems more suitable to describe the disease pathophysiology, given also that major early and late clinical complications (i.e. CV and chronic hydrocephalus) spatially correlate best with this compartment. Many of the included proteins have previously been associated to SAH inflammation, for example IL-1ra, IL-6, IL-8, TNF-a, LIF, MCP-1, and VEGF-A.[32] On the other hand, there is scarce or non-existing literature on many other proteins, some of which showed interesting temporal patterns and statistically significant peaks and trends, such as LIF, CCL11, CCL28, 4E-BP1, CD40, CXCL6, CXCL9, and IL-18.[20, 33–36]
Group A
Nine biomarkers showed higher day 1 values and decreasing trends throughout the observation period (Tables 3 and 4). Four of them (CCL11, LIF, MIP-1β and TRAIL) showed statistically significant early peaks with day 1 > day 4 levels but no statistically significant decreasing trends were observed. MIP-1β (also known as CCL4) and CCL11 are members of the chemokine family (C-C motif) and are involved in chemotaxis of macrophages and activated T-cells, respectively, as well as other proinflammatory actions. Their early peak can possibly be associated to the recruitment of leukocytes at the site of the bleeding. LIF is a cytokine involved in activation of signaling pathways that regulate cell growth among other actions. A similar early peak in serum has been observed previously.[37] TRAIL, a member of the TNF superfamily (also known as TNFSF10) involved in apoptosis, has not been studied in the SAH literature.
Group B
Among the 14 proteins included in this group only two (CCL28 and DNER) showed statistically significant middle peaks, that is significantly higher day 4 values than both day 1 and day 10. Production of the chemokine CCL28 is induced by other proinflammatory cytokines and its chemotactic actions are exerted on B and T cells and eosinophils. Delta and Notch-like Epidermal growth factor-Receptor (DNER) is an activator of NOTCH1 pathway. None of these biomarkers have been studied in a SAH context earlier and their potential involvement in the SAH complications, mainly CV that coincides temporally with the observed middle peaks, should be examined.
It should be noted that the majority of the biomarkers in this group showed a significant increase in their levels between day 1 and day 4 (11/14, Table 4). In almost all cases though, with the exception of the two biomarkers named above, these dynamics seemed to wear off between day 4 and day 10. Interestingly, IL-6 was recently studied by our group using a quantitative routine monoclonal antibody-based method in CSF samples from 44 patients with severe SAH. The results showed a very similar temporal IL-6 pattern as in the present study, with increasing values between day 1 and day 4 followed by decreasing values towards day 10, although remaining higher than day 1 values.[38]
Group C
Half of the studied biomarkers (n = 27) showed higher levels towards the end of the observation period, reflecting a more prolonged activation post SAH that may indicate an involvement in the healing processes or the development of late complications, such as late vasospasm, posthemorrhagic hydrocephalus, etc. Fifteen biomarkers (4E-BP1, CASP-8, CCL19, CCL23, DC40, CDCP1, CXCL1, CXCL6, CXCL9, IL18, IL8, MCP-2, OPG, STAMPB and uPA) showed statistically significant late peaks, meaning significantly higher day 10 than day 4 values. Apart from CASP-8, IL-18, IL-8, OPG and STAMPB all the remaining proteins in the list showed statistically significant increasing trends throughout the observation period with day 1 < day 4 < day 10 values.
Chemokines CCL23, CXCL6 and CXCL9, all potent chemotactic agents for resting T-cells/monocytes, neutrophilic granulocytes and T-cells respectively, have not been studied in SAH patients earlier. Signaling pathway molecule 4E-BP1 has been implicated in the development of vasospasm in a canine SAH-model but no reports on human studied are available.[35] Protein CD40, a member of TNF family, is found on antigen-presenting cells and mediates multiple inflammatory responses. Elevated serum levels of CD40 have been associated with poor outcome and severity of neurological deficits in SAH patients.[36, 39] IL-18, a strong proinflammatory cytokine involved in the synthesis of inflammatory mediators, has recently been shown to be a predictor of early brain injury and clinical prognosis in SAH patients as elevated concentrations correlated to cerebral edema and acute hydrocephalus.[20] It should be noted that the observed temporal pattern of IL-18 in that study differed from our study as we demonstrated a late peak of this cytokine. Urokinase (or uPA) is a serum protease that activates plasminogen to plasmin which in turn leads to thrombolysis and tissue degradation. Plasma concentrations of its receptor (soluble uPA-receptor) was not shown to correlate with neurological outcome post SAH.[40]
General considerations
The great variability of the expression levels and temporal patterns of the measured biomarkers is an indicator of the complexity of the inflammatory response after SAH. Many of the included proteins are well established in the SAH research both in humans and in preclinical animal models while others are novel in the SAH field. Their exact role as well as interplay with each other is not easy to establish, especially considering the fact that many of these substances are described to play both a detrimental and a beneficial role in the disease course depending on the time after bleeding.[41] This study may offer guidance for further research on groups of biomarkers based either on their families or their pattern of activation (e.g. early vs late peak) to identify potential underlying inflammatory mechanisms of SAH complications as well as novel targets of intervention in order to prevent/treat these conditions and improve outcome.
Limitations
A limitation with the study is that the PEA analysis method in its presently available form does not give absolute protein concentrations. However, the protein expression levels illustrate the relative concentrations and how these change over time. They also illustrate the relations between the levels of the different biomarkers. The study may be limited by the relatively small number of patients included. Another drawback may be the fact that the study is limited to the CSF compartment. Similar analyses could be performed in the CSF, cerebral interstitial fluid and plasma, giving the opportunity to compare protein levels in the different fluid compartments providing a more thorough inflammatory profile of the acute phase of the disease. Comparisons with healthy individuals could also serve as an indicator of the intensity of activation for each biomarker. Moreover, correlations of the biomarker expression levels with clinical parameters were not performed in this study, as the main goal was to provide general information of as many proteins as possible in order to look for patterns of expression for further investigation. Finally, measurements in more than three time points could possibly describe the temporal patterns of each protein more accurately.