Cerebrospinal Fluid and Microdialysis Cytokines in Aneurysmal Subarachnoid Hemorrhage: A Scoping Systematic Review

Objective To perform two scoping systematic reviews of the literature on cytokine measurement in cerebral microdialysis (CMD) and cerebrospinal fluid (CSF) in aneurysmal subarachnoid hemorrhage (SAH) patients, aiming to summarize the evidence relating cytokine levels to pathophysiology, disease progression, and outcome. Methods Two separate systematic reviews were conducted: one for CMD cytokines and the second for CSF cytokines. Data sources Articles from MEDLINE, BIOSIS, EMBASE, Global Health, Scopus, Cochrane Library (inception to October 2016), reference lists of relevant articles, and gray literature were searched. Study selection Two reviewers independently identified all manuscripts utilizing predefined inclusion/exclusion criteria. A two-tier filter of references was conducted. Data extraction Patient demographic and study data were extracted to tables. Results There were 9 studies identified describing the analysis of cytokines via CMD in 246 aneurysmal SAH patients. Similarly, 20 studies were identified describing the analysis of CSF cytokines in 630 patients. The two scoping systematic reviews demonstrated the following: (1) limited literature available on CMD cytokine measurement in aneurysmal SAH with some preliminary data supporting feasibility of measurement and potential association between interleukin (IL)-6 and patient outcome. (2) Various CSF measured cytokines may be associated with patient outcome at 3–6 months, including IL-1ra, IL-6, IL-8, and tumor necrosis factor-alpha. (3) There is a small literature body supporting an association between acute/subacute CSF transforming growth factor levels and the development of chronic hydrocephalus at 2–3 months. Conclusion The evaluation of CMD and CSF cytokines is an emerging area of the literature in aneurysmal SAH. Further large prospective multicenter studies on cytokines in CMD and CSF need to be conducted.

iNTRODUCTiON Inflammation in the setting of aneurysmal subarachnoid hem orrhage (SAH) is believed to be a potential driver of many secondary insults in this often critically ill population (1)(2)(3)(4). As with animal models of stroke, inflammatory mediators have been associated with loss of salvageable ischemic penumbra, infarct propagation, and cerebral edema (5)(6)(7)(8). It has been proposed that various inflammatory cytokines are associated with secondary injury and pathological processes postaneurysmal SAH (1,2). Within the aneurysmal SAH population, numerous studies have been published associating serum inflammatory markers with patient outcome and the risk of cerebral vasospasm/delayed ischemic neurological deficits (DINDs) (1,3,4). Furthermore, analysis of cerebrospinal fluid (CSF) cytokines has demonstrated an association between interleukin (IL)6 and tumor necrosis factoralpha (TNFa) with the risk of cerebral vasospasm and DIND through an extensive systematic review/metaanalysis of the available literature (9). Of note, this systematic review focused only on the relationship IL6 and TNFa with vasospasm, with out any comment or consideration of other important primary outcomes such as patient morbidity/mortality.
The goal of this project was to produce a scoping systematic review of the literature on both CMD and CSF cytokines in aneurysmal SAH. There were two main focuses for this article: (1) to provide a comprehensive scoping systematic review of the literature on CMD cytokines in aneurysmal SAH; (2) produce a scoping systematic review evaluating the association between CSF cytokines and the following outcomes (excluding vasos pasm/DIND): patient functional outcome, neurophysiologic outcome, chronic hydrocephalus/VSP dependency, and tissue outcome.

MeTHODS
Two separate scoping systematic reviews were conducted, using the methodology outlined in the Cochrane Handbook for Systematic Reviewers (38). Data were reported following the Preferred Reporting Items for Systematic Reviews and Meta Analyses (39). The review questions and search strategy were decided upon by the primary author (Frederick A. Zeiler) and supervisors (Adel Helmy and David K. Menon).
This article was conducted in concert with a similar review on cytokines in CMD and CSF for severe traumatic brain injury (TBI) patients, which is currently unpublished and under review (40).

Search Question and Population of interest
Given that two separate systematic reviews were conducted, one for CMD cytokines and the other for CSFbased cytokines, two distinct questions were posed. The lack of literature identified through a preliminary search of PubMed led us to conduct a scoping review for the CMD cytokine search, with the attempt to identify all studies in this area to date. The larger literature base for CSF cytokines in aneurysmal SAH led us to narrow our ques tion for this scoping review, focusing on relevant outcomes (see below). The questions posed for this scoping systematic review were as follows: 1. What literature has been published on CMD of cytokines in aneurysmal SAH? 2. Is there literature to suggest an association between CSFbased cytokine measures in aneurysmal SAH and patient outcome, chronic hydrocephalus/shunt dependency, neurophysiologic outcome, or tissue outcome?
For the CMD cytokine scoping review, all articles describ ing microdialysisbased cytokine measures in humans with aneurysmal SAH were included to provide a comprehensive overview.
For the CSF cytokine review, the primary outcome measures of interest were documented association between CSF meas ured cytokines and patient outcome, chronic hydrocephalus/ shunt dependency, neurophysiologic outcome (as measured via intensive care unit (ICU)based monitoring, intracranial pressure/cerebral perfusion pressure, brain tissue oxygen monitoring (PbtO2), thermal diffusion assessment of cerebral blood flow (CBF), transcranial Doppler (TCD) measure of cerebral blood flow velocity, any neuroimagingbased assess ment of CBF/perfusion, and electrophysiology), and tissue outcome [as assessed on followup neuroimaging by either computed tomography (CT) or magnetic resonance imaging]. Any outcome score or mention of morbidity/mortality within the studies was deemed acceptable for documentation of patient outcome. Secondary outcome measures were complica tions associated with CSF monitoring of cytokines. Of note, cerebral vasospasm and DIND were specifically excluded as a primary outcome for the CSF cytokine review given a recently conducted systematic review published on this exact relation ship (9). Acceptable cytokines in CMD or CSF included IL1a, IL1b ,  IL1ra, IL2, sIL2ra, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10,  IL11, IL12, IL12p70, IL13, IL14, IL15, IL16, IL17, inducible  protein10, eotaxin, TNF, interferon gamma, monocyte chemoat tractant proteins (MCP), macrophage inflammatory proteins (MIPs), transforming growth factor (TGF), nerve growth factor, brainderived neurotrophic factor, glialderived neurotrophic factor, soluble tumor necrosis factor receptor (sTNFR), granu locyte macrophage colonystimulating factor (GMCSF), sFAS, soluble vascular cell adhesion molecule1, soluble intracellular adhesion molecule1, plateletderived growth factor, RANTES, macrophagederived chemokine (MDC), Flt3, fractalkine, and fibroblast growth factor receptor. In addition, we surveyed relevant meeting proceedings for the last 5 years looking for ongoing and unpublished work based on the cytokine analysis via CMD or CSF in aneurysmal SAH patients. We elected to include published meeting proceedings to provide as comprehensive of a scoping systematic review as possible. It is acknowledged that the quality of evidence derived from such pseudopeerreviewed meeting publications is limited. However, given that cytokine research in SAH is relatively "new" and our goal was to produce a systematically conducted scoping review on the topic, we elected to include them to be comprehensive. The meeting proceedings of the following profes sional societies were searched: Finally, reference lists of any review articles on CSF or CMD cytokines were reviewed for any missed relevant studies.

Study Selection
Utilizing two reviewers, a twostep review of all articles returned by our search strategies was performed. First, the reviewers inde pendently (Frederick A. Zeiler and Eric Peter Thelin) screened titles and abstracts of the returned articles to decide if they met the inclusion criteria. Second, full text of the chosen articles was then assessed to confirm if it met the inclusion criteria and that the primary outcome of interest was reported in the studies (Frederick A. Zeiler and Eric Peter Thelin). Any discrepancies between the two reviewers were resolved by a third reviewer if needed (Adel Helmy or David K. Menon).

Data Collection
Data were extracted from the selected articles and stored in an electronic database. Data fields included type of study, article location, number of patients, patient demographics, aneurysm characteristics/treatment, Hunt and Hess (H + H) clinical grade (41), World Federation of Neurological Surgeons (WFNS) clinical grade (42), Fisher CT grade (43), ICU therapies applied, CMD/CSF substrate measured, CMD/CSF measurement details (probe tissue location and sampling frequency), outcome meas ure described (patient, neurophysiologic, and tissue), association between CMD/CSF cytokine measure to outcome, and compli cations. Complications of interest for the CSF studies were any related to ventriculostomy: misplacement, tract hemorrhage, infection, and extraaxial hemorrhage/collection formation. All data for both the CSF and CMD cytokine studies can be found in Tables 1-4.

Bias Assessment
As the goal of this study was to produce a systematically con ducted scoping review of the available literature on CMD and CSF cytokine measures in aneurysmal SAH, formal bias assess ment was not done. Our desire was to produce a comprehensive overview of the current literature on the topic of CMD/CSF cytokines in aneurysmal SAH. Formal evidence grading was not conducted (given the limited and heterogenous literature body), and thus, we deemed formal bias risk assessment unnecessary for this emerging area of literature, which clearly suffers from standard biases associated with new areas of clinical research.

Statistical Analysis
A metaanalysis was not performed in this study due to the heterogeneity of data and study design within the articles identified.
Secondary: none mentioned Fisher CT score: aSAH Primary: to determine the association between CSF TGF-b1 and TGF-b2 with shunt dependency post-aSAH Admission WFNS: Secondary: to compare CSF cytokines in aSAH and non-hemorrhage hydrocephalus patients Admission Fisher CT score: Aneurysm location: Secondary: none mentioned Admission Fisher score: Aneurysm characteristics: aSAH Primary: to determine the association between CSF IL-6 and vasospasm Admission WFNS: Secondary: none mentioned Admission Fisher CT Score: Aneurysm Location: Secondary: to evaluate patient outcome Admission Fisher CT score: Aneurysm location:

CMD Cytokine Search
Search strategy results for CMD cytokines in aneurysmal SAH can be seen within the flow diagram in Figure 1. At total of 60 references were returned, all coming from the database search and none identified via meeting proceeding searches. After duplicate removal, there were 30 articles left for the assessment via the first filtering of title and abstract content. Thirteen articles passed the first filter, requiring acquisition of the full manuscript to assess inclusion eligibility. Through assessment of the full articles, nine manuscripts were deemed eligible for inclusion in the final CMD systematic review. No articles were added from the reference sections of either review papers or the parent manuscripts included in the systematic review.

CSF Cytokine Search
The search strategy flow diagram for the CSF cytokine scoping systematic review can be seen in Figure 2. Overall, 516 articles were identified, with 513 from the database search and 3 from published meeting proceedings. Two hundred and eighty dupli cates removed, leaving 236 references to review in the first filter. After implementation of the first filter, 61 articles were selected for assessment of their full manuscripts. One additional reference was added from the reference sections of review papers. After the second filter of full manuscripts, 20 articles were deemed eli gible for final inclusion in the CSF systematic review. Remaining articles were excluded due to nonrelevance.

CDM Cytokine Review
All of the nine articles included in the CMD cytokine portion of the systematic review all were formal manuscript publications        (10)(11)(12)(13)(14)(15)(16)(17)(18). There were six prospective studies, with all being pro spective observational studies (10,11,(13)(14)(15)18). Three studies were retrospective case series (12,16,17). A total of 246 unique patients with SAH were described across the 9 studies included in the CMD cytokine review. Two studies reported the same group of 88 SAH CMD patients, with a focus on analyzing different cytokine measures (15,16). We took this into account during the calculation of the total patient numbers, to avoid counting patients twice.
The patient populations described within the CMD cytokine manuscripts were heterogeneous collections of aneurysmal SAH. The majority of studies focused on patients with poor admission clinical grades, classified as Hunt and Hess (H + H) grade 3-5, or as World Federation of Neurological Surgeons (WFNS) grade 3-5; often with high Fisher CT grade hemorrhages (i.e., 3 or 4). Aneurysm locations varied between studies, with some included both anterior and posterior circulation aneurysms. When recorded, the highest percentage of aneurysms was located within the anterior circulation with: anterior communicating artery (AComm), middle cerebral artery (MCA), and posterior com municating artery (PComm) locations predominating. Three studies failed to disclose patient clinical grade, radiographic grade, and aneurysm location information (14)(15)(16). Therapies received in the ICU were not clearly specified in the majority of the studies. Similarly, aneurysm treatment technique varied between and within the individual studies, with microsurgical clipping predominating.
We believe that some of the studies included within this por tion of the review may contain duplicate patient information, as marked in Tables 1 and 3. Multiple publications from the same research groups likely were conducted on the same patient popu lations, yielding unique and separate manuscripts on the same group of patients. However, it is important to acknowledge that it was difficult to determine, in some circumstances, whether CMD cytokine analysis was being conducted on new patient groups or existing banked samples from previous prospective studies. With that said, our goal for the CMD cytokine scoping review was to provide an overview of all available literature in the area; hence, we have included all published papers on CMD cytokines in aneurysmal SAH within this review.
The populations described with in the CSF cytokine studies were quite heterogeneous, similar to the CMD cytokine papers. Most studies focused on patients with poor clinical grade (i.e., H + H 3-5; WFNS 3-5), and high Fisher CT grade (i.e., 3 or 4) upon admission. Aneurysm location varied significantly between papers with both anterior and posterior circulation aneurysms included in the studies. The majority of patients had anterior circulation aneurysms with AComm, MCA, and PComm rep resenting the three most common locations. Therapies received within the ICU were either not specified or minimally character ized, leading to potentially significant treatment heterogeneity during CSF cytokine measurement. Finally, aneurysm treatment technique was unspecified in many studies (10,19,22,25,28,30,31,(33)(34)(35)37). Those studies which disclosed aneurysm treat ment employed both coiling and microsurgical clipping (20,21,23,24,26,27,29,32,36).
A total of 630 patients were described across all studies included in the CSF cytokine systematic review. The mean age for each study cohort varied significantly across studies, with all studies focusing on adult aneurysmal SAH. Details surrounding patient cohort, study design, and concurrent therapies can be found in Tables 2 and 4.
We made substantial efforts to exclude duplicate patient data across studies. However, given that many of the papers came from centers of excellence for TBI research, some of the patient data may be cross reported in multiple studies. This would reduce the total overall number of unique patients slightly. It was impossible based on the information provided within the parent studies to tease out all patients, which were reported more than once.

CMD Cytokine Review
Location of the CMD catheter varied significantly between stud ies and was the following: mixed healthy/perilesional tissue in six studies (13)(14)(15)(16)18), territory of the aneurysm in two studies (10,17), and unknown tissue location in two studies (11,13). Some studies utilized paired microdialysis catheters, one in healthy and one in perilesional tissue (15,16).
Analysis interval for CMD samples was as follows: every 6 h in five studies (11,12,(14)(15)(16), every 8-12 h in one study (17), and unspecified in three studies (10,13,18). The duration of sample collection varied as well, with the typical collection period of 36 h to 10 days.
Numerous different panels of cytokines were evaluated within the CMD samples, across the studies included within the review. The most commonly studied cytokines included IL1b, IL6, IL10, and TNFa. Details surrounding CMD technique and catheter locations can be seen in Table 3.
Like the CMD cytokine papers, the CSF cytokine papers included in this review reported the measurement of various cytokines. The most commonly measured cytokines in CSF reported were IL1b, IL1ra, IL6, IL8, TNF, TGF, and MIP. The details of CSF sampling and specific cytokines measured can be found in Table 4.

CMD Cytokine Review
Given that the CMD cytokine portion of this review was a scop ing review, providing an overview of all the available literature on CMD cytokine measures in aneurysmal SAH, the outcomes reported by the studies are quite heterogenous. They can be seen in detail in Table 3.
Three studies reported the correlation between CMD cytokines and patient outcome (10,13,18). All of these three studies reported a correlation between elevated CMD IL6 levels and poor outcome at 6 months, measured using the Glasgow Outcome Scale (GOS) (10), 3 months measured using the modi fied Rankin scale (mRS) (p = 0.01) (13), or other unspecified outcome scales (p < 0.01) (18).
Similarly, perilesional probe CMD probe location was associated with higher IL6 levels compared to more distant probe locations (p = 0.002) (18).
Radiographic cerebral vasospasm was found to be associated with elevated CMD TNFa on day 4 and 6 postictus in one study (p < 0.01) (12). Similarly, elevated total CMD IL6 levels were found to be associated with radiographic vasospasm in one study (17). However, there was an unclear association with the develop ment of DIND.
Many studies provided descriptions of CMD cytokine profiles and temporal patterns. Given the various cytokines measured across the studies, it is impossible to describe all of these relation ships, but highlights from these analyses are presented in Table 3.
Broadly speaking, the data show temporal variations in cytokine levels, with peaks in IL1b, IL6, IL8, and MIP between 6 and 12 h postbleed (14,15). On the other hand, IL10 levels in CMD remained constantly elevated throughout the analysis periods recorded (14,15).
Chronic Hydrocephalus/VPS Dependency. Four studies dis cussed the correlation between CSF cytokines and the devel opment of chronic hydrocephalus/VPS dependency (28)(29)(30)(31). One study showed that CSF TGFb1 and TGFb2 levels were associated with the development of hydrocephalus (defined using CT imaging) at 2 months postbleed (p < 0.05) (28). A second study also documented the correlation between elevated CSF TGFb1 during the patients ICU stay and the development of radiographic hydrocephalus or VPS dependency at 3 months (p < 0.02) (29). A third study confirmed that CSF TGFb1 lev els were elevated during the acute/subacute phase in those who became shunt dependent (30). Finally, one study documented that CSF IL6 levels during the acute/subacute phase postbleed to be associated with VPS dependency at an unclear interval (p = 0.009) (31).
Two studies reported no association between CSF cytokine measures and TCDbased flow velocity (32,36), while one study failed to show an association between CSF TGF and VPS depend ency (33). Further detail on the "nil association" studies can be found at the bottom of Table 4.

Complications
Within the CMD cytokine manuscripts, all manuscripts failed to report whether complications were considered within the data collection. We suspect that complication profiles are dramatically underreported within the CMD studies.
Complication reporting within the CSF cytokine studies was essentially nonexistent, with the focus of these studies being the association between CSF cytokine measures and various outcomes.

CMD Cytokines in SAH
The scoping systematic review on CMD cytokines in aneurysmal SAH yielded nine studies. Despite the small number of studies and patients described within, there are a few points of interest that deserve highlighting. First, CMDbased measurement of cytokines is feasible in this patient population. Second, CMD catheter location makes a difference in the levels of cytokines measured, with perilesional tissue producing high levels com pared to distant or healthier tissue (18). Third, peaks in CMD cytokine measures may occur within the first 6-12 h for IL1b, IL6, IL8 and, MIP, while IL10 seems to remain elevated in CMD samples through the duration of the sampling periods described (14,15). Fourth, CMD IL6 levels may be associated with poor outcome (10,13,18), up to 6 months postinjury. Finally, com plications related to the use of CMD catheters are underreported, and there is a concern of selective harms reporting within the literature identified.
Limitations Despite the interesting results of these two systematic reviews, there are significant study limitations that need to be highlighted. Limitations with each separate review can be found within the subsections to follow. Two limitations affected both reviews. First, which was eluded to within the Methods section, is the inclusion of meeting abstracts. This could be considered contro versial; however, to provide the most comprehensive scoping sys tematic review on this relatively "new" field of research in SAH, we thought it necessary to include these studies. Furthermore, many negative studies are presented at meeting venues, never reaching manuscript form. We wished to include any of these potential negative result abstracts to reduce publication bias seen within only positive studies. Yet, one must be cautioned in overinterpreting the results of the meeting abstracts. Given the nature of these publications, the quality of evidence is low and they are subject to significant reporting biases.
Second, within both the CMD and CSF reviews, some stud ies had missing data points, as seen within the tables. We made two distinct and separate attempts to contact the authors for information regarding these studies (i.e., missing demograph ics, etc.). The first was made in November 2016, with a second attempt in January 2017. Both were met with no response via electronic communication. Thus, we were unfortunately left with leaving these fields as "unknown" or "uncertain" within the tables. Although this is unfortunate, as the overall picture for each study may not be complete, this tends to be the nature of systematically conducted reviews within new and emerging areas of research.
Finally, the exact details on the cytokine measurements were not clearly delineated in most studies. With little comment on what was done to reduce interassay variability, as this could contribute to conflicting results seen within the review. Some mentioned use an enzymelinked immunosorbent assay (ELISA) for the cytokine(s) of interest, while others mentioned multiplex "plates" for and array of cytokines, without further details. Furthermore, the timing of cytokine measurement was not mentioned or taken into consideration the reported studies. Thus, there is potential for normal circadian variation in cytokine profiles to have impacted the results reported.

CMD Cytokine Review
First, there were a small number of heterogenous studies found for the CMD review, with some manuscripts reporting on the same patient populations based on banked CMD samples. Furthermore, all included studies described heterogeneous cohorts of aneurysmal SAH patients with varying clinical/ radiographic admission grades and aneurysm locations, making summary interpretation of results difficult. Second, the ICU and surgical therapies received by these patients during CMD sample collection/processing was quite heterogeneous. Many studies failed to specify the therapies or protocols initialized within the ICU. These treatment variations may lead to substantial changes within the CMD cytokine measures. Thus, the described associa tions or "nil associations" may not be accurate given this potential confounder. Third, across all the studies, there was variation in CMD catheter location. This could impact the CMD cytokine measures obtained and the described relationships. In addition, the CMD perfusate, sampling frequency, use of pooled analysis, and cytokine panel/analytic platform employed varied between studies. Given this, it is impossible for us to directly compare the absolute values of cytokines and relative recovery. Thus, our reporting of the results for CMD in SAH is limited to purely descriptive. Fourth, complications associated with CMD moni toring were seldom reported. Given the total number of patients studied, it is unlikely that there were no patients suffering from complications of invasive monitoring. Finally, given the studies and results identified for the CMD review, there is likely a large publication bias, favoring only studies with positive results.

CSF Cytokine Review
First, there were many quite heterogeneous studies identified in the CSF cytokine review. The included papers varied by number of patients, admission clinical/radiographic grades, aneurysm location, aneurysm treatments (clipping vs. coiling), surgical interventions, ICUbased therapies offered/provided to patients, blinding during outcome assessment, primary outcome of the studies, and duration of followup. These limitations suggest caution when interpreting or generalizing the results of studies that describe relationships between CSF cytokine measures and patient outcomes. Second, many cytokine associations were selectively reported, making no reference to other CSF measures and the results of statistical analysis. Therefore, there may be many more "nil associations" that were not disclosed within the body of the manuscripts. Third, complication reporting was concerning within the literature identified (as mentioned above), with under reporting is suspected. Fourth, given all the above limitations and heterogeneity issues, a metaanalysis was not performed. Finally, given the overwhelming number of "positive association" studies identified, the literature likely suffers from significant publication bias.

CMD Technical Considerations
The complexity involved in cytokine retrieval from CMD requires some brief comments regarding some potentially more "stand ardized" techniques. First, standard CMD catheters employ pore sizes between 20 and 100 kDa, as the goal with these devices is to measure "common" analytes such as glucose, glutamate, glycerol, lactate, and pyruvate. Although well designed for this purpose, they are ineffective for the retrieval or larger protein biomark ers, such as cytokines, where molecular weight can easily exceed these pore size. This it is critical to know the characteristics of the biomarker of interest, thus tailoring your CMD catheter to the biomarker (44). Second, the location of placement is key. Within the TBI literature, it has been well documented that CMD catheter placement in lesional vs. perilesional tissue yields very different profiles of "common" analyte retrieval (45). This has also been demonstrated within CMD cytokine profiles in TBI, with lesional/perilesional tissue expressing much high cytokine levels compared to healthy tissue (46,47). Thus, we recommend place ment of the CMD catheter within the brain adjacent to a focal lesion or territory of interest. This way, the "at risk" brain would be monitored and not the irreversibly damaged areas. Third, the rate of perfusion should remain at 0.3μl/min. Higher perfu sion rates may impair the rate of uptake of these larger proteins (44), while there is not data to support improved recovery for lower rates. Fifth, the perfusate should be colloid based. Recent investigation into the type of perfusate has demonstrated that the relative rate of recovery for cytokines is improved with colloid perfusate over crystalloid (44). The exact colloid solution to use is currently unclear. Albumin solution appears to improve the rela tive recovery (44); however it is expensive and labor intensive to create, thus limiting its widespread applicability. Dextranbased solutions are another potential and have been applied within some of the SAH studies quoted within this review (14,16). However, the literature surrounding the type of dextran solution to use is limited, and we cannot make any further definitive comments at this time. Sixth, it is unclear at the current time as to the impact of frequency of CMD measurement and pooling of samples. Given we do not currently have a clear idea of the temporal profile of cytokine changes in CMD fluid, we cannot give definitive recom mendations regarding the sampling frequency. Although, we would expect the rate of change in focal cytokine profile be on the order of hours or longer (such as 6 to 12 h). Finally, the cytokine analysis technique requires some comment and is applicable to both CMD and CSF analysis. Both ELISA and multiplexbased techniques have been described. All techniques are subject to interassay variability and thus should be conducted within estab lished laboratory settings with trained personal, comfortable with the employed technique. The use of ELISA vs. multiplex may be site dependent or study specific. Standard analytic techniques should be employed for multicenter collaborations to improved homogeneity in measurement technique. This also applied to the entire process of CMD or CSF sampling, storage, and analysis.
In addition, the normal variation in cytokine profiles should be taken into account when determining sampling frequency and pooling samples for analysis. It is particularly important to ensure that between patients, the sampling frequency and employment of sample pooling is conducted in an identical manner. Circadian variation in cytokine profiles (48) could impact the interpreta tion of results, and thus, standardized sampling and pooling is a necessity.

Future Directions
Given the limited literature body and the recognized limitations in study design, there exists an opportunity for further research in the area of CMD and CSF cytokines in aneurysmal SAH, but these should seek to address the limitations seen in the studies included within the two systematic reviews. First, larger cohorts of aneurysmal SAH patients with predefined stratification of injury pattern are required. Heterogeneity in hemorrhage pat tern, clinical grade, and aneurysm location make the results of the abovementioned studies difficult to interpret, even those with positive results. Large sample sizes may allow for clinical/ radiographic subgroup analysis and shed further light on the association of CMD/CSF cytokines with various subpopulations of aneurysmal SAH patients. While large studies undertaken with a uniform protocol would be ideal, we need to accept that these studies will often be conducted in relatively small populations of patients across several centers. Such a multiplicity of studies could be a substantial strength in exploring the pathophysiology and outcome associations of central nervous system cytokine levels across the spectrum of aSAH if we could undertake harmoniza tion of the studies. Consistency across multiple centers would require rigorous harmonization of studies, which would only be possible if there were clear data provided on disease character istics, catheter location, sample processing, and measurement techniques; and all studies used a common outcome assessment (e.g., GOSE at 3 months). With the application of common data elements between studies and centers, we may be able to more closely approach harmonization. Furthermore, banking of CSF and CMD samples from various SAH studies could prove to be a useful way of increasing the sample numbers required to analyze the milieu of CSF/CMD based cytokines. Second, homogeneous ICU/surgical treatments are necessary, preferably with protocol ized therapies. Including coiled and clipped patients within the same cohort of SAH patients assuredly confounds the associa tions between various measured cytokines and the described out comes. In addition, including patients with ICH evacuation and those undergoing DC due to malignant edema will also impact the resulting of cytokine measures. Third, with the application of CMD, catheter location must be considered during cytokine measures. Fourth, given the large number of cytokines involved, the use of principle component analysis of large patient popula tions with CMD and CSF cytokine measures may prove valuable. This has been applied within the TBI literature on CMD cytokines, with interesting preliminary results (49,50). This could poten tially identify cytokine patterns of coexpression in CMD and CSF, highlighting targets for future studies and therapeutic inter vention. Fifth, accurate complication documentation is required. Sixth, one persistent problem may be the use of different analysis platforms, which results in different measured concentrations. There are no easy solutions to this problem-although control plasma levels will provide some basis for harmonization, it will be difficult to get standardize levels in CSF and particularly in CMD. Finally, multicenter prospective evaluation of cytokines within CMD and CSF is necessary to improve patient recruitment and aid with spreading the substantial cost of cytokine analysis among centers. Without collaboration, singlecenter small studies may unfortunately fail to add to the existing literature.

CONCLUSiON
The evaluation of CMD and CSF cytokines is a new area of the literature in aneurysmal SAH. The two scoping systematic reviews demonstrated the following: (1) limited literature avail able on CMD cytokine measurement in aneurysmal SAH with some preliminary data supporting feasibility of measurement and potential association between IL6 and patient outcome. (2) CSF levels of several cytokines may be associated with patient outcome at 3-6 months including IL1ra, IL6, IL8, and TNFa.
(3) There is a small literature supporting an association between acute/subacute CSF TGF levels and the development of chronic hydrocephalus at 2-3 months. Given the preliminary nature of these data, further large prospective multicenter studies on cytokines in CMD and CSF need to be conducted.

AUTHOR CONTRiBUTiONS
FZ was responsible for concept, design, systematic review searches, data acquisition/extraction, data analysis, manuscript composition, and editing. ET was responsible for systematic review searches, data acquisition/extraction, data analysis, manuscript composition, and editing. MC was responsible for data analysis, manuscript composition, and editing. PH was responsible for data analysis and manuscript composition/edit ing. DM and AH were responsible for concept, data analysis, and manuscript composition/editing.