Megakaryocytes respond during sepsis and display innate immune cell behaviors.

Megakaryocytes (MKs) are precursors to platelets, the second most abundant cells in the peripheral circulation. However, while platelets are known to participate in immune responses and play significant functions during infections, the role of MKs within the immune system remains largely unexplored. Histological studies of sepsis patients identified increased nucleated CD61+ cells (MKs) in the lungs, and CD61+ staining (likely platelets within microthrombi) in the kidneys, which correlated with the development of organ dysfunction. Detailed imaging cytometry of peripheral blood from patients with sepsis found significantly higher MK counts, which we predict would likely be misclassified by automated hematology analyzers as leukocytes. Utilizing in vitro techniques, we show that both stem cell derived MKs (SC MKs) and cells from the human megakaryoblastic leukemia cell line, Meg-01, undergo chemotaxis, interact with bacteria, and are capable of releasing chromatin webs in response to various pathogenic stimuli. Together, our observations suggest that MK cells display some basic innate immune cell behaviors and may actively respond and play functional roles in the pathophysiology of sepsis.

A PREVENTIVE TOOL FOR PREDICTING BLOODSTREAM INFECTIONS IN CHILDREN WITH BURNS.

ABSTRACT: Introduction: Despite significant advances in pediatric burn care, bloodstream infections (BSIs) remain a compelling challenge during recovery. A personalized medicine approach for accurate prediction of BSIs before they occur would contribute to prevention efforts and improve patient outcomes. Methods: We analyzed the blood transcriptome of severely burned (total burn surface area [TBSA] ≥20%) patients in the multicenter Inflammation and Host Response to Injury ("Glue Grant") cohort. Our study included 82 pediatric (aged <16 years) patients, with blood samples at least 3 days before the observed BSI episode. We applied the least absolute shrinkage and selection operator (LASSO) machine-learning algorithm to select a panel of biomarkers predictive of BSI outcome. Results: We developed a panel of 10 probe sets corresponding to six annotated genes ( ARG2 [ arginase 2 ], CPT1A [ carnitine palmitoyltransferase 1A ], FYB [ FYN binding protein ], ITCH [ itchy E3 ubiquitin protein ligase ], MACF1 [ microtubule actin crosslinking factor 1 ], and SSH2 [ slingshot protein phosphatase 2 ]), two uncharacterized ( LOC101928635 , LOC101929599 ), and two unannotated regions. Our multibiomarker panel model yielded highly accurate prediction (area under the receiver operating characteristic curve, 0.938; 95% confidence interval [CI], 0.881-0.981) compared with models with TBSA (0.708; 95% CI, 0.588-0.824) or TBSA and inhalation injury status (0.792; 95% CI, 0.676-0.892). A model combining the multibiomarker panel with TBSA and inhalation injury status further improved prediction (0.978; 95% CI, 0.941-1.000). Conclusions: The multibiomarker panel model yielded a highly accurate prediction of BSIs before their onset. Knowing patients' risk profile early will guide clinicians to take rapid preventive measures for limiting infections, promote antibiotic stewardship that may aid in alleviating the current antibiotic resistance crisis, shorten hospital length of stay and burden on health care resources, reduce health care costs, and significantly improve patients' outcomes. In addition, the biomarkers' identity and molecular functions may contribute to developing novel preventive interventions.

Physical Rehabilitation and Mental Health Care After Burn Injury: A Multinational Study.

While remarkable improvements have been made to acute hospital burn care in recent decades, it is not matched by improvements in post-acute care, including physical rehabilitation and mental health. Progress in acute hospital treatment of burn survivors now highlights the next important step-addressing care once a patient leaves intensive treatment and is discharged to the community. Long-term physical rehabilitation and mental health services are vital to improving quality of life for burn survivors. Using qualitative methods, we apply an adapted Reeve framework to assess and compare post-acute physical rehabilitation and mental health care across 13 countries on 6 continents. Twenty semistructured interviews were conducted with burn surgeons and rehabilitation specialists. One major theme that emerged was the importance of training and resources to the quality of post-acute care. This exploratory study suggests the value of investing scarce resources in a range of low-cost interventions to improve follow-up burn care. One intervention identified here is short-term training in post-acute rehabilitation and mental health to upgrade and standardize best clinical practices to address as-yet unmet post-discharge needs of burn survivors.

Transcriptomic responses from improved murine sepsis models can better mimic human surgical sepsis.

Historically, murine models of inflammation in biomedical research have been shown to minimally correlate with genomic expression patterns from blood leukocytes in humans. In 2019, our laboratory reported an improved surgical sepsis model of cecal ligation and puncture (CLP) that provides additional daily chronic stress (DCS), as well as adhering to the Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) guidelines. This model phenotypically recapitulates the persistent inflammation, immunosuppression, and catabolism syndrome observed in adult human surgical sepsis survivors. Whether these phenotypic similarities between septic humans and mice are replicated at the circulating blood leukocyte transcriptome has not been demonstrated. Our analysis, in contrast with previous findings, demonstrated that genome-wide expression in our new murine model more closely approximated human surgical sepsis patients, particularly in the more chronic phases of sepsis. Importantly, our new model of murine surgical sepsis with chronic stress did not reflect well gene expression patterns from humans with community-acquired sepsis. Our work indicates that improved preclinical murine sepsis modeling can better replicate both the phenotypic and transcriptomic responses to surgical sepsis, but cannot be extrapolated to other sepsis etiologies. Importantly, these improved models can be a useful adjunct to human-focused and artificial intelligence-based forms of research in order to improve septic patients' morbidity and mortality.

Multi-Biomarker Prediction Models for Multiple Infection Episodes Following Blunt Trauma.

Severe trauma predisposes patients to multiple independent infection episodes (MIIEs), leading to augmented morbidity and mortality. We developed a method to identify increased MIIE risk before clinical signs appear, which is fundamentally different from existing approaches entailing infections' detection after their establishment. Applying machine learning algorithms to genome-wide transcriptome data from 128 adult blunt trauma patients' (42 MIIE cases and 85 non-cases) leukocytes collected ≤48 hr of injury and ≥3 days before any infection, we constructed a 15-transcript and a 26-transcript multi-biomarker panel model with the least absolute shrinkage and selection operator (LASSO) and Elastic Net, respectively, which accurately predicted MIIE (Area Under Receiver Operating Characteristics Curve [AUROC] [95% confidence intervals, CI]: 0.90 [0.84-0.96] and 0.92 [0.86-0.96]) and significantly outperformed clinical models. Gene Ontology and network analyses found various pathways to be relevant. External validation found our model to be generalizable. Our unique precision medicine approach can be applied to a wide range of patient populations and outcomes.

Denver and Marshall scores successfully predict susceptibility to multiple independent infections in trauma patients.

Trauma patients are at risk of repeated hospital-acquired infections, however predictive scores aiming to identify susceptibility to such infections are lacking. The objective of this study was to investigate whether commonly employed disease-severity scores can successfully predict susceptibility to multiple independent infectious episodes (MIIEs) among trauma patients. A secondary analysis of data derived from the prospective, longitudinal study "Inflammation and the Host Response to Injury" ("Glue Grant") was performed. 1,665 trauma patients, older than 16, were included. Patients who died within seven days from the time of injury were excluded. Five commonly used disease-severity scores [Denver, Marshall, Acute Physiology and Chronic Health Evaluation II (APACHE II), Injury Severity Score (ISS), and New Injury Severity Score (NISS)] were examined as independent predictors of susceptibility to MIIEs. The latter was defined as two or more independent infectious episodes during the index hospital stay. Multivariable logistic regression was used for the statistical analysis. 22.58% of the population was found to be susceptible to MIIEs. Denver and Marshall scores were highly predictive of the MIIE status. For every 1-unit increase in the Denver or the Marshall score, there was a respective 15% (Odds Ratio:1.15; 95% CI: 1.07-1.24; p < 0.001) or 16% (Odds Ratio:1.16; 95% CI: 1.09-1.24; p < 0.001) increase in the odds of MIIE occurrence. APACHE II, ISS, and NISS were not independent predictors of susceptibility to MIIEs. In conclusion, the Denver and Marshall scores can reliably predict which trauma patients are prone to MIIEs, prior to any clinical sign of infection. Early identification of these individuals would potentially allow the implementation of rapid, personalized, preventative measures, thus improving patient outcomes and reducing healthcare costs.

Megakaryocytes contain extranuclear histones and may be a source of platelet-associated histones during sepsis.

Histones are typically located within the intracellular compartment, and more specifically, within the nucleus. When histones are located within the extracellular compartment, they change roles and become damage-associated molecular patterns (DAMPs), promoting inflammation and coagulation. Patients with sepsis have increased levels of extracellular histones, which have been shown to correlate with poor prognosis and the development of sepsis-related sequelae, such as end-organ damage. Until now, neutrophils were assumed to be the primary source of circulating histones during sepsis. In this paper, we show that megakaryocytes contain extranuclear histones and transfer histones to their platelet progeny. Upon examination of isolated platelets from patients with sepsis, we identified that patients with sepsis have increased amounts of platelet-associated histones (PAHs), which appear to be correlated with the type of infection. Taken together, these results suggest that megakaryocytes and platelets may be a source of circulating histones during sepsis and should be further explored.

Translation and Cross-cultural Validation of the English Young Adult Burn Outcome Questionnaire (YABOQ) in Spanish.

The Young Adult Burn Outcome Questionnaire (YABOQ) is a validated, English-language patient-reported outcome assessment of young adults' recovery from burn injury across 15 scale domains. We evaluated the cross-cultural validity of a newly developed Spanish version of the YABOQ. Secondary data from English- and Spanish-speaking burn survivors (17 to 30 years of age) were obtained from the Multicenter Benchmarking Study. We conducted classic psychometric analyses and evaluated the measurement equivalence of the English and Spanish YABOQs in logistic and ordinal logistic regression differential item functioning analyses. All multi-item scales in the Spanish YABOQ demonstrated adequate reliability except the Pain and Itch scales. One item in the Perceived Appearance scale showed differential item functioning across English- and Spanish-speaking burn survivors, but the observed differential item functioning had no clinically significant impact on scale-level Perceived Appearance scores. Our findings support the cross-cultural validity of the YABOQ Physical Function, Perceived Appearance, Sexual Function, Emotion, Family Function, Family Concern, Satisfaction with Symptom Relief, Satisfaction with Role, Work Reintegration and Religion scales among English- and Spanish-speaking young adult burn survivors. This work supports the use of these English and Spanish YABOQ scales to assess the effect of therapeutic interventions on young adults' burn outcomes in pooled analyses and to assess disparities in young adults' burn outcomes across language groups.

Genome-wide comparisons of gene expression in adult versus elderly burn patients.

PURPOSE: Mortality and morbidity rates of elderly burn patients remain high despite numerous advancements in modern burn care. While prior studies have offered first insights on the biochemical changes in elderly burn patients compared to adults, the underlying cellular responses remain largely unknown. In this study, we aim to characterize the transcriptome of elderly burn patients and compare it to adult burn patients to obtain insights into the underlying molecular responses post-burn and to elucidate the effect of advanced age on the acute burn response. MATERIALS AND METHODS: Microarray data obtained from the Glue Grant Trauma-Related Database was obtained from blood specimens for ten elderly patients (n = 10), each with a set of two sex and total body surface area (TBSA) matched adult controls (n = 20), during the acute phase post-burn. Adult and elderly demographics and clinical outcomes were contrasted using using the Chi-Square test, Fisher's Exact Test, or two-sample t-tests, as appropriate (p<0.05). Enrichment and heat maps were generated to compare gene expression in elderly versus adult burn patients. RESULTS: Supervised analysis identified multiple genes that were differentially expressed between the elderly and adult groups. Pathway analysis and heatmap generation suggest that elderly patients share a distinct hypo-inflammatory response in the acute post-burn phase with downregulation of a number of immune-related pathways, including those related to antigen processing, specifically via MHC class I, ubiquitination and proteasome degradation (p<0.001, FDR < .001). Cell signalling pathways, such as NF-κB, C-type lectin receptor, and T cell receptor signalling were also significantly downregulated in elderly burn patients, as well as those relating to antiviral immunity (p<0.001, FDR < .001). Many genes which were observed to be upregulated in elderly patients with high TBSA burn injuries were associated with destruction-related cellular pathways such as complement activation and immunoglobulin production (p<0.005, FDR <0.01). CONCLUSIONS: The altered inflammatory and immune responses at the transcriptome level in elderly patients after burn are indicative of a failure in elderly burn patients to initiate an appropriate inflammatory and stress response during the acute phase post-burn.

Associations between clinical characteristics and the development of multiple organ failure after severe burns in adult patients.

To determine the association between potential risk factors and multiple organ failure (MOF) in severe burn adult patients, we performed a secondary analysis of data from the "Inflammation and the Host Response to Injury" database, which included patients from six burn centers in the United States between 2003 and 2009. Three hundred twenty-two adult patients (aged ≥16 years) with severe burns (≥20.0% total body surface area [TBSA]) were included. MOF was defined according to the Denver score. Potential risk factors were analyzed for their association with MOF. Models were built using multivariable logistic regression analysis. Eighty-eight patients (27.3%) developed MOF during the study period. We found that TBSA, age, and inhalation injury were significant risk factors for MOF. This predictive model showed good performance, with the total area under the receiver operating characteristic curve being 0.823. Moreover, among patients who developed MOF, inhalation injury was significantly associated with the development of MOF in the acute phase (within three days of injury) (adjusted odds ratio 3.1; 95% confidence interval 1.1-8.3). TBSA, age, lactate, and Denver score within 24h were associated with the late phase development of MOF. Thus, we have identified key risk factors for the onset of MOF after severe burn injury. Our findings contribute to the understanding of individualized treatment and will potentially allow for efficient allocation of resources and a lower threshold for admission to an intensive care unit, which can prevent the development of MOF and eventually reduce mortality.

Developing Effective Alzheimer's Disease Therapies: Clinical Experience and Future Directions.

Alzheimer's disease (AD) clinical trials, focused on disease modifying drugs and conducted in patients with mild to moderate AD, as well as prodromal (early) AD, have failed to reach efficacy endpoints in improving cognitive function in most cases to date or have been terminated due to adverse events. Drugs that have reached clinical stage were reviewed using web resources (such as clinicaltrials.gov, alzforum.org, company press releases, and peer reviewed literature) to identify late stage (Phase II and Phase III) efficacy clinical trials and summarize reasons for their failure. For each drug, only the latest clinical trials and ongoing trials that aimed at improving cognitive function were included in the analysis. Here we highlight the potential reasons that have hindered clinical success, including clinical trial design and choice of outcome measures, heterogeneity of patient populations, difficulties in diagnosing and staging the disease, drug design, mechanism of action, and toxicity related to the long-term use. We review and suggest approaches for AD clinical trial design aimed at improving our ability to identify novel therapies for this devastating disease.

Coenzyme Q10 protects against burn-induced mitochondrial dysfunction and impaired insulin signaling in mouse skeletal muscle.

Mitochondrial dysfunction is associated with metabolic alterations in various disease states, including major trauma (e.g., burn injury). Metabolic derangements, including muscle insulin resistance and hyperlactatemia, are a clinically significant complication of major trauma. Coenzyme Q10 (CoQ10) is an essential cofactor for mitochondrial electron transport, and its reduced form acts as a lipophilic antioxidant. Here, we report that burn injury induces impaired muscle insulin signaling, hyperlactatemia, mitochondrial dysfunction (as indicated by suppressed mitochondrial oxygen consumption rates), morphological alterations of the mitochondria (e. g., enlargement, and loss of cristae structure), mitochondrial oxidative stress, and disruption of mitochondrial integrity (as reflected by increased mitochondrial DNA levels in the cytosol and circulation). All of these alterations were significantly alleviated by CoQ10 treatment compared with vehicle alone. These findings indicate that CoQ10 treatment is efficacious in protecting against mitochondrial dysfunction and insulin resistance in skeletal muscle of burned mice. Our data highlight CoQ10 as a potential new strategy to prevent mitochondrial damage and metabolic dysfunction in burn patients.

A New Test for the Detection of Direct Oral Anticoagulants (Rivaroxaban and Apixaban) in the Emergency Room Setting.

Determining whether a patient has taken a direct oral anticoagulant (DOAC) is critical during the periprocedural and preoperative period in the emergency department. However, the inaccessibility of complete medical records, along with the generally inconsistent sensitivity of conventional coagulation tests to these drugs, complicates clinical decision making and puts patients at risk of uncontrollable bleeding. In this study, we evaluate the utility of inhibitor-II-X (i-II-X), a novel, microfluidics-based diagnostic assay for the detection and identification of Factor Xa inhibitors (FXa-Is) in an acute care setting. DESIGN: First-in-human, 91-patient, single-center retrospective pilot study. SETTING: Emergency room. PATIENTS: Adult patients admitted into the emergency department, which received any clinician-ordered coagulation test requiring a 3.2% buffered sodium citrate blood collection tube. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma samples from patients admitted to the emergency department were screened for the use of FXa-Is, including apixaban and rivaroxaban, within the past 24 hours using our new i-II-X microfluidic test. i-II-X results were then compared with results from conventional coagulation tests, including prothrombin time (PT) and international normalized ratio (INR), which were ordered by treating clinicians, and an anti-Xa assay for rivaroxaban. The i-II-X test detected DOACs in samples collected from the emergency department with 95.20% sensitivity and 100.00% specificity. Unlike PT and INR, i-II-X reliably identified patients who had prolonged clotting times secondary to the presence of a FXa-I. CONCLUSIONS: The i-II-X test overcomes the limitations of currently available coagulation tests and could be a useful tool by which to routinely screen patients for DOACs in emergency and critical care settings. Our new diagnostic approach is particularly relevant in clinical situations where medical records may be unavailable, or where precautions need to be taken prior to invasive interventions, such as specific reversal agent administration.

The Effect of Facial Burns on Long-Term Outcomes in Young Adults: A 5-Year Study.

Long-term functional outcomes in young adults with facial burns remain poorly studied. This 5-year (2003-2008) prospective multicenter study includes burn survivors (age 19-30 years) who completed the Young Adult Burn Outcome Questionnaire (YABOQ) from 0 to 36 months after baseline survey administration. A composite canonical score was developed from 15 YABOQ domains using discriminant analysis, maximizing the difference at the baseline between burn-injured patients with face involved and not involved. A generalized linear model with the generalized estimation equation technique was used to track the changing pattern of the composite score over time. Individual domain scores with high correlation to the canonical score were used to evaluate recovery patterns in facial burns. A total of 153 burned (31% with face burns) and 112 nonburned subjects completed 620 questionnaires. Canonical analysis showed that early postburn, facial burns were associated with a difference in outcome, but this overall difference diminished over time. Regression analysis showed that for survivors with facial injury, Emotion and Sexual Function scores were persistently lower (worse), while Religion scores were persistently higher. Satisfaction with Role was initially better than the nonface burned group, but over time got worse, while Perceived Appearance was initially worse in the face burned group but this difference diminished over time. Social Function Limited by Appearance was initially similar between the groups, but over time the group with face burns scored lower. The overall difference in recovery between survivors with and without facial burns diminished over time while the individual domains had various patterns of recovery.

Development of clinical process measures for pediatric burn care: Understanding variation in practice patterns.

BACKGROUND: There has been little systematic examination of variation in pediatric burn care clinical practices and its effect on outcomes. As a first step, current clinical care processes need to be operationally defined. The highly specialized burn care units of the Shriners Hospitals for Children system present an opportunity to describe the processes of care. The aim of this study was to develop a set of process-based measures for pediatric burn care and examine adherence to them by providers in a cohort of pediatric burn patients. METHODS: We conducted a systematic literature review to compile a set of process-based indicators. These measures were refined by an expert panel of burn care providers, yielding 36 process-based indicators in four clinical areas: initial evaluation and resuscitation, acute excisional surgery and critical care, psychosocial and pain control, and reconstruction and aftercare. We assessed variability in adherence to the indicators in a cohort of 1,076 children with burns at four regional pediatric burn programs in the Shriners Hospital system. The percentages of the cohort at each of the four sites were as follows: Boston, 20.8%; Cincinnati, 21.1%; Galveston, 36.0%; and Sacramento, 22.1%. The cohort included children who received care between 2006 and 2010. RESULTS: Adherence to the process indicators varied both across sites and by clinical area. Adherence was lowest for the clinical areas of acute excisional surgery and critical care, with a range of 35% to 48% across sites, followed by initial evaluation and resuscitation (range, 34%-60%). In contrast, the clinical areas of psychosocial and pain control and reconstruction and aftercare had relatively high adherence across sites, with ranges of 62% to 93% and 71% to 87%, respectively. Of the 36 process indicators, 89% differed significantly in adherence between clinical sites (p < 0.05). Acute excisional surgery and critical care exhibited the most variability. CONCLUSION: The development of this set of process-based measures represents an important step in the assessment of clinical practice in pediatric burn care. Substantial variation was observed in practices of pediatric burn care. However, further research is needed to link these process-based measures to clinical outcomes. LEVEL OF EVIDENCE: Therapeutic/care management, level IV.

Burn-induced muscle metabolic derangements and mitochondrial dysfunction are associated with activation of HIF-1α and mTORC1: Role of protein farnesylation.

Metabolic derangements are a clinically significant complication of major trauma (e.g., burn injury) and include various aspects of metabolism, such as insulin resistance, muscle wasting, mitochondrial dysfunction and hyperlactatemia. Nonetheless, the molecular pathogenesis and the relation between these diverse metabolic alterations are poorly understood. We have previously shown that burn increases farnesyltransferase (FTase) expression and protein farnesylation and that FTase inhibitor (FTI) prevents burn-induced hyperlactatemia, insulin resistance, and increased proteolysis in mouse skeletal muscle. In this study, we found that burn injury activated mTORC1 and hypoxia-inducible factor (HIF)-1α, which paralleled dysfunction, morphological alterations (i.e., enlargement, partial loss of cristae structure) and impairment of respiratory supercomplex assembly of the mitochondria, and ER stress. FTI reversed or ameliorated all of these alterations in burned mice. These findings indicate that these burn-induced changes, which encompass various aspects of metabolism, may be linked to one another and require protein farnesylation. Our results provide evidence of involvement of the mTORC1-HIF-1α pathway in burn-induced metabolic derangements. Our study identifies protein farnesylation as a potential hub of the signaling network affecting multiple aspects of metabolic alterations after burn injury and as a novel potential molecular target to improve the clinical outcome of severely burned patients.

Adult-onset, chronic, cyclic thrombocytopenia in a Rhesus macaque (Macaca mulatta) after dengue virus vaccination and viral challenge.

An 8-year-old, male Rhesus macaque (Macaca mulatta), previously used for dengue virus (DENV) vaccine research with viral challenge, was presented with adult-onset, chronic, cyclic thrombocytopenia. Platelet number, morphology, and function were evaluated by automated hematology, peripheral blood smears, electron microscopy, flow cytometry, and impedance aggregometry. Bone marrow was evaluated by cytology. Both serum anti-dengue nonstructural protein 1 (NS1) antibodies and anti-platelet antibodies were detected by ELISA. Platelet characterization showed a lack of aggregation to all agonists (ADP, ASP, and collagen), increased activation with increased expression of surface marker (HLA-ABC), and an absence of surface receptor GPIX during clinical episodes of petechiae and ecchymoses, even in the presence of normal platelet counts. Bone marrow aspirates identified potential mild megakaryocytic hypoplasia. All platelet functions and morphologic attributes were within normal limits during clinically normal phases. Presence of anti-dengue NS1 serum antibodies confirmed a positive DENV titer 8 years postvaccination. Based on the history and clinical findings, a primary differential diagnosis for this chronic, cyclic platelet pathology was autoimmune platelet destruction with potential bone marrow involvement.

Local and Systemic Changes Associated with Long-term, Percutaneous, Static Implantation of Titanium Alloys in Rhesus Macaques (Macaca mulatta).

Metal alloys are frequently used as implant materials in veterinary medicine. Recent studies suggest that many alloys induce both local and systemic inflammatory responses. In this study, 37 rhesus macaques with long-term skull-anchored percutaneous titanium alloy implants (duration, 0 to 14 y) were evaluated for changes in their hematology, coagulation, and serum chemistry profiles. Negative controls (n = 28) did not have implants. Macaques with implants had higher plasma D-dimer and lower antithrombin III concentrations than nonimplanted animals. In addition, animals with implants had higher globulin and lower albumin and calcium concentrations compared with nonimplanted macaques. Many of these changes were positively correlated with duration of implantation and the number of implants. Chronic bacterial infection of the skin was present around many of the implant sites and within deeper tissues. Representative histopathology around the implant site of 2 macaques revealed chronic suppurative to pyogranulomatous inflammation extending from the skin to the dura mater. X-ray fluorescence microscopy of tissue biopsies from the implant site of the same 2 animals revealed significantly higher levels of free metal ions in the tissue, including titanium and iron. The higher levels of free metal ions persisted in the tissues for as long as 6 mo after explantation. These results suggest that long-term skull-anchored percutaneous titanium alloy implants can be associated with localized inflammation, chronic infection, and leaching of metal ions into local tissues.