Perioperative cerebrospinal fluid and plasma inflammatory markers after orthopedic surgery

Cognitive impairment, including the conditions of postoperative delirium [1, 2] and postoperative cognitive dysfunction (POCD) [2, 3], is frequently observed after major surgery, particularly in older patients [3‐7]. Postoperative delirium is associated with increases in length of hospital stay, admissions to long-term care institutions, long-term functional decline, and long-term mortality [8]. POCD, which is measured by cognitive testing, has been similarly shown to be associated with premature inability to work, dependency on social transfer payments, inability to cope independently, and long-term mortality [7, 9]. Although risk factors such as advanced age and neurodegenerative processes have been identified [5], the pathophysiology for either condition is still unclear.

Orthopedic procedures may result in extensive trauma, tissue injury, and blood loss, which can engage the innate immune system [10]. Preclinical studies suggest that inflammation is a possible pathogenic mechanism for POCD [11‐15]. In particular, increased expression of cytokines in a rodent’s hippocampus following surgery was associated with cognitive decline [12, 16, 17]. Putative mediators of this postoperative neuroinflammatory response include interleukin (IL)-1β [12], IL-6, tumor necrosis factor-α (TNF-α), and high-mobility group box 1 protein (HMGB1) [16, 18].

Some studies suggest that surgical patients also exhibit elevations of proinflammatory cytokines in the central nervous system and systemic circulation [19‐23]. However, three of these studies were performed in patients who had sustained traumatic injury prior to surgery; thus, the preceding trauma may have contributed to inflammation [19‐21]. One study was performed in patients who underwent correction of idiopathic nasal cerebrospinal fluid (CSF) leak [24]. In this setting, the perforation of the blood-brain barrier due to the underlying pathology may have led to biomarker increases in the CSF that cannot be clearly differentiated from the changes induced by surgery. Another research team evaluated biomarkers in patients who received concurrent continuous infusion of bupivacaine into the spinal space through the same indwelling spinal catheter that was used for sampling [22, 23, 25, 26]. Given bupivacaine’s anti-inflammatory properties and potential association with chemical meningitis [27, 28], the reported results may have been confounded by this concurrent administration [29‐31].

To address the potential relationship between surgery, systemic and central inflammatory biomarkers, and postoperative cognitive changes, we designed a prospective cohort pilot study. The study was performed in patients undergoing elective major knee surgery, a clinical equivalent of our previous preclinical model of aseptic surgery [11‐13].

We hypothesized that aseptic surgery resulted in systemic inflammation and inflammation of the central nervous system. Results from this study will enable us to design future larger studies to test the hypothesis that persistent and excessive neuroinflammation caused by aseptic surgery triggers postoperative cognitive changes.

Recruitment is summarized in a summary and flow diagram in Additional file 2. A total of 141 patients were asked to participate in the study between 12/2010 and 4/2013. Sixty-nine patients declined, 29 patients were ineligible due to medical conditions, and 17 for other reasons. Eight patients were removed after starting the study (causes explained in Additional file 2). Sampling was performed without complications in ten patients; this cohort forms the basis of the report. Nine patients underwent total knee arthroplasty, and one patient had a knee extensor tendon reconstruction with allograft. The mean age of the patients was 70.3 (8.7) years, range 59–84 years. The average blood loss was 105 (16) ml, average tourniquet time was 21 (21) min, and bone cement was used in 9/10 patients. At 24 h after surgery, the catheter was removed in all patients. No patient developed complications related to the study procedure. CSF and plasma cytokine analysis was performed in all ten patients. Demographics and comorbidities are summarized in Table 1 (patient characteristics). Data for the patients that underwent baseline cognitive testing, but were ultimately not included in the study, are provided in Additional file 3c.

We observed substantial elevations in proinflammatory cytokines in both plasma and CSF after aseptic surgery (Tables 2 and 3). The pronounced changes in CSF cytokines compared to plasma for several cytokines (MCP, MIP-1α, MIP-1β) provide evidence for substantial inflammatory activity in the central nervous system. In particular, the statistically significant increases in plasma IL-5, IL-6, IL-8, MCP-1, MIP-1α, and RAGE and of anti-inflammatory IL-10 suggest a substantial activation of key pathways of the immune response. The activation of CSF IL-5, IL-6, IL-8, MCP-1, MIP-1α, and MIP-1β indicates that chemoattraction of monocytes may play a key role similar to what had been previously observed in preclinical studies [14, 18, 41]. Moreover, the cytokine pattern changes suggest an immunological response that includes B-cell stimulation, immunoglobulin secretion, activation of T-cells, eosinophil and neutrophil granulocytes, and simultaneous activation of anti-inflammatory pathways. Release of mitochondrial damage-associated molecular patterns from femur fracture reamings also activate neutrophils, which release IL-8 and MMP9 that contribute to remote organ injury [42, 43].

Due to its sample size, this was a feasibility study with predominantly descriptive findings. Our results cannot determine any causal association of cytokine changes with postoperative cognitive changes. However, there were marginally significant differences in the postoperative changes of plasma IL-5 and IL-8 from baseline in patients with and without postoperative cognitive changes (Fig. 2 and Table 2). This result provides a starting point for future research.

In addition, our preliminary results show that proinflammatory plasma cytokines were substantially elevated in the patient who developed delirium. Moreover, a persistent increase in proinflammatory cytokines IL-6, IL-8, and MCP-1 was observed in the CSF of this patient. It is noteworthy that this patient, while meeting criteria for elective surgery, was among the older patients in our cohort and among these with more comorbidities. It remains to be proven whether preoperative subclinical inflammation may be a predictor for postoperative delirium.

Patients who developed POCD had decreased levels of several anti- and proinflammatory cytokines. Equally, our findings for POCD are limited by the size of the dataset and the relatively discrete signs of POCD that were observed in 60 % of the patients. Further studies are needed to confirm these alterations.

Based on our results, an important consideration for future work may be the altered levels of several cytokines at baseline in patients who later developed POCD or delirium. Currently, the significance of these alterations is unclear and needs to be investigated in future studies with larger sample size. Research to further elucidate these alterations may be useful to identify patients who are at risk for postoperative surgical changes. However, while significant correlations between plasma and CSF samples were present for IL-6, IL-8, MIP-1α, and MIP-1β, correlations were negligible for other cytokines. This is a potentially important result of our work, since it may indicate limits to the utility of measurements of some cytokines in more readily available plasma samples, and the potentially usefulness of this approach for others. Our results support the conclusions from a recent meta-analysis [44] that found a correlation between POCD and the peripheral markers of IL-6 and S100β [44] but indicate limited applicability of plasma approaches for other cytokines.

The scientific context of our results contains animal studies that demonstrate that surgical stimulus activates the immune system even under aseptic conditions [10]. Several preclinical studies have established inflammation of the central nervous system as a pathogenic mechanism for cognitive dysfunction [12‐15, 45]. Release of proinflammatory mediators including damage-associated molecular patterns (DAMPs), such as HMGB1, into the circulation have been shown to impair the blood-brain barrier permeability after orthopedic surgery in mice [14]. These cytokines converge into activation of the innate immune system by convergence on NF-кB pathways. In the circulation, HMGB1 interacts with pattern recognition receptors (toll-like receptors 2 and 4 as well as receptor for advanced glycation end products) on immunocytes. Through a permeable blood-brain barrier, peripheral monocyte-derived macrophages access the brain parenchyma. These are attracted by signaling from the hippocampal MCP-1, a chemokine that regulates migration and infiltration of C–C motif receptor 2-expressing cells [41, 46, 47]. These processes have been associated with memory dysfunction in preclinical models of POCD, and strategies to block macrophage infiltration and/or excessive proinflammatory mediators have been successful in reducing these memory deficits [14, 48]. Macrophage-specific IкB kinase (IKK)β coordinates activation of NF-кB; when it is deleted, it prevents the blood-brain barrier disruption and infiltration of bone marrow-derived macrophages into the hippocampus following surgery [48]. Learning and memory processes rely on the hippocampus. Rodent studies have shown that increased expression of cytokines and DAMPs in rodent’s hippocampus following surgery was associated with cognitive decline [12, 16, 17]. Potential mediators identified in animal models include IL-1β [12], IL-6, TNF-α, and HMGB1 [16, 18]. Existing clinical studies show accumulating evidence that the CSF’s isolation from inflammatory reactions (immune privilege) [49] may not be sustained perioperatively. Baune et al. [50] reported a correlation between plasma IL-8 levels and cognitive performance in 369 community-dwelling elderly subjects. Beloosesky and coworkers [19] investigated the functional status and plasma cytokine levels in 41 hip fracture patients. They reported significantly higher plasma C-reactive protein and IL-6 kinetics curves in patients with preoperatively impaired mental status. Recent studies in patients undergoing emergency surgery reported that preoperative inflammatory markers were elevated in plasma and CSF [20] [21] and that the extent of this inflammatory reaction might correlate with the degree of postoperative cognitive decline [20]. In these studies, the cause of the observed biomarker increase cannot be clearly attributed to surgery.

The change in CSF/plasma albumin ratio after aseptic peripheral surgery [22] indicates an alteration in the blood-brain barrier permeability in the perioperative period. When evaluating plasma and CSF samples for up to 30 h postoperatively, a consistent upregulation of CSF IL-6, a transient increase in CSF IL-8, a delayed increase in PGE2, and undetectable IL-1β, TNF-α, and IL-10 in CSF [25] were found.

Several investigations have been performed in one patient population [22, 23, 25, 26]. Bromander et al. [23] investigated changes in CSF and serum in 35 patients undergoing knee arthroplasty surgery preoperatively, 3 and 24 h postoperatively. It was determined that IL-2, IL-8, IL-10, and IL-13 were increased to more than 500 % of their initial concentrations, while TNF content doubled [23]. However, the concurrent administration of bupivacaine in these studies may have affected the reported results. Bupivacaine has known anti-inflammatory properties [29‐31, 51] and has been associated with chemical meningitis [27, 28]. Of note, patients in our study group received a single dose of bupivacaine at the time of placement of the catheter with no subsequent administrations.

Tang [24] measured cytokines in 11 patients undergoing endoscopic surgery to correct idiopathic nasal CSF drip under general anesthesia. Mean CSF Aβ-42 remained unchanged, but total-tau and phosphorylated-tau181P increased progressively until 48 h after surgery. CSF IL-10, S100β, IL-6, and TNF-α were increased at 24 h. Their results may indicate neuronal damage and/or aggravation of existing conditions. However, the underlying CSF rhinorrhea [24] implies a breach of the blood-brain barrier, potentially causing direct immune activation in the CSF, and meningitis is a potential complication [52]. In addition, the condition is frequently associated with intracranial hypertension [53], and fluorescein was injected via the lumbar catheter into the CSF during this surgery [54]. Cape and coworkers [55] assessed 43 patients aged >60 years with acute hip fracture for delirium before and 3–4 days after surgery. CSF samples were taken at the induction of spinal anesthesia. Delirium was diagnosed in eight patients before surgery; 17 patients developed delirium after surgery. The authors found that CSF IL-1β was significantly higher in patients with new-onset delirium compared to patients without delirium and that CSF to serum IL-1β ratios were higher in delirious than non-delirious patients. CSF IL-1ra was significantly higher in patients with delirium before surgery compared to new-onset delirium.

In the context of the above literature, our study is the first to demonstrate significant perioperative changes in a wide range of inflammatory markers in a setting without trauma and with minimal injury to the cerebrospinal space. Interestingly, these findings are more pronounced in CSF than in plasma, which could indicate that they originate from the central nervous system.

There are, however, several potential limitations to our study. We used CAM to determine the presence of postoperative delirium. CAM was developed as a screening instrument for delirium but has high sensitivity and specificity. When compared with the delirium rating scale, which has ten features, CAM measured 9/10 features except for physical disorder [56]. Whether using the delirium rating scale will yield different results will need to be evaluated by future studies. In addition, the follow-up period limits our data on POCD. Third, because of the small sample size, our study results will need validation by a larger study. We included a broader age range of patients in order to optimize patient recruitment. The inclusion of older subjects may result in a higher incidence of postoperative delirium or POCD. Additional considerations include more frequent CSF and plasma sampling over a more extended period to consider other than the effect of circadian variations [57]. Moreover, it is currently unknown to what extent intravenous sedation [58] and a single bolus injection of bupivacaine for spinal anesthesia affects cytokine levels in CSF and plasma. An inflammatory response as a result of the in-dwelling catheter cannot be excluded from our data. Preoperative medications, pain, and use of opioids and other drugs postoperatively may as well affect cognitive status. Lastly, idiosyncrasies of the surgical procedure [59, 60], such as tourniquet use [60, 61, 62] reaming of the femur [42, 43] with potential for fat emboli, and cementing of the prosthesis [63], may influence the results. Tourniquet times were 21.0 (20.7) min. Its role as a cofactor in this study is probably small, since cytokine patterns in the two patients with the longest tourniquet time (about 60 min) were not discernible from the remaining patients. Similarly, there was no difference in cytokine patterns in the patient that did not undergo reaming of the femur and cementing (data not shown).

In summary, our results demonstrate increased inflammatory activity in plasma and CSF in the perioperative period and shed more light on the complex inflammatory mechanisms occurring during and after surgery. These results corroborate previous studies [23, 25] that a proinflammatory response based on IL-6 and IL-8 is present in CSF and, less markedly, in plasma after aseptic surgery. In addition, we found elevated levels of the HMGB1 receptor RAGE, consistent with previously reported elevated levels of HMGB1 [16, 18, 64] and TNF-α [13, 23] in both animal and clinical studies. Moreover, our results and previous work [23] establish a role for the anti-inflammatory cytokine IL-10. The role for IL-1β requires further clarification; while elevated levels were found in animal studies [12], we and others [25] were unable to reproduce this finding after aseptic surgery in humans. Further studies need to elucidate the crosstalk between systemic markers of inflammation and those present in the CSF. While we found significant alterations in some cytokines and substantial alterations in others in plasma and CSF from patients with postoperative cognitive changes, further research is needed to elucidate potential contributions to the pathogenesis of postoperative delirium and POCD.