Accuracy of head computed tomography scoring systems in predicting outcomes for patients with moderate to severe traumatic brain injury: A ProTECT III ancillary study.

BACKGROUND: Several types of head CT classification systems have been developed to prognosticate and stratify TBI patients. OBJECTIVE: The purpose of our study was to compare the predictive value and accuracy of the different CT scoring systems, including the Marshall, Rotterdam, Stockholm, Helsinki, and NIRIS systems, to inform specific patient management actions, using the ProTECT III population of patients with moderate to severe acute traumatic brain injury (TBI). METHODS: We used the data collected in the patients with moderate to severe (GCS score of 4-12) TBI enrolled in the ProTECT III clinical trial. ProTECT III was a NIH-funded, prospective, multicenter, randomized, double-blind, placebo-controlled clinical trial designed to determine the efficacy of early administration of IV progesterone. The CT scoring systems listed above were applied to the baseline CT scans obtained in the trial. We assessed the predictive accuracy of these scoring systems with respect to Glasgow Outcome Scale-Extended at 6 months, disability rating scale score, and mortality. RESULTS: A total of 882 subjects were enrolled in ProTECT III. Worse scores for each head CT scoring systems were highly correlated with unfavorable outcome, disability outcome, and mortality. The NIRIS classification was more strongly correlated than the Stockholm and Rotterdam CT scores, followed by the Helsinki and Marshall CT classification. The highest correlation was observed between NIRIS and mortality (estimated odds ratios of 4.83). CONCLUSION: All scores were highly associated with 6-month unfavorable, disability and mortality outcomes. NIRIS was also accurate in predicting TBI patients' management and disposition.

Comparing blood biomarkers to clinical decision rules to select patients suspected of traumatic brain injury for head computed tomography.

INTRODUCTION: Traumatic brain injury (TBI) is a major public health concern in the U.S. Recommendations for patients admitted in the emergency department (ED) to receive head computed tomography (CT) scan are currently guided by various clinical decision rules. OBJECTIVE: To compare how a blood biomarker approach compares with clinical decision rules in terms of predicting a positive head CT in adult patients suspected of TBI. METHODS: We retrospectively identified patients transported to our emergency department and underwent a noncontrast head CT due to suspicion of TBI and who had blood samples available. Published thresholds for serum and plasma glial fibrillary acidic protein (GFAP), ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1), and serum S100ß were used to make CT recommendations. These blood biomarker-based recommendations were compared to those achieved under widely used clinical head CT decision rules (Canadian, New Orleans, NEXUS II, and ACEP Clinical Policy). RESULTS: Our study included 463 patients, of which 122 (26.3%) had one or more abnormalities presenting on head CT. Individual blood biomarkers achieved high negative predictive value (NPV) for abnormal head CT findings (88%-98%), although positive predictive value (PPV) was consistently low (25%-42%). A composite biomarker-based decision rule (GFAP+UCH-L1)'s NPV of 100% and PPV of 29% were comparable or better than those achieved under the clinical decision rules. CONCLUSION: Blood biomarkers perform at least as well as clinical rules in terms of selecting TBI patients for head CT and may be easier to implement in the clinical setting. A prospective study is necessary to validate this approach.

Association between Blood and Computed Tomographic Imaging Biomarkers in a Cohort of Mild Traumatic Brain Injury Patients.

The objective of this work was to analyze the relationships between traumatic brain injury (TBI) on computed tomographic (CT) imaging and blood concentration of glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase-L1 (UCH-L1), and S100B. This prospective cohort study involved 644 TBI patients referred to Stanford Hospital's Emergency Department between November 2015 and April 2017. Plasma and serum samples of 462 patients were analyzed for levels of GFAP, UCH-L1, and S100B. Glial neuronal ratio (GNR) was calculated as the ratio between GFAP and UCH-L1 concentrations. Admission head CT scans were reviewed for TBI imaging common data elements, and performance of biomarkers for identifying TBI was assessed via area under the receiver operating characteristic curve (ROC). We also dichotomized biomarkers at established thresholds and estimated standard measures of classification accuracy. We assessed the ability of GFAP, UCH-L1, and GNR to discriminate small and large/diffuse lesions based on CT imaging using an ROC analysis. In our cohort of mostly mild TBI patients, GFAP was significantly more accurate in detecting all types of acute brain injuries than UCH-L1 in terms of area under the curve (AUC) values (p < 0.001), and also compared with S100B (p < 0.001). UCH-L1 and S100B had similar performance (comparable AUC values, p = 0.342). Sensitivity exceeded 0.8 for each biomarker across all different types of TBI injuries, and no significant differences were observed by type of injury. There was a significant difference between GFAP and GNR in distinguishing between small lesions and large/diffuse lesions in all injuries (p = 0.004, p = 0.007). In conclusion, GFAP, UCH-L1, and S100B show high sensitivity and negative predictive values for all types of TBI lesions on head CT. A combination of negative blood biomarkers (GFAP and UCH-L1) in a patient suspected of TBI may be used to safely obviate the need for a head CT scan. GFAP is a promising indicator to discriminate between small and large/diffuse TBI lesions.

Blood Biomarkers for Detection of Brain Injury in COVID-19 Patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus attacks multiple organs of coronavirus disease 2019 (COVID-19) patients, including the brain. There are worldwide descriptions of neurological deficits in COVID-19 patients. Central nervous system (CNS) symptoms can be present early in the course of the disease. As many as 55% of hospitalized COVID-19 patients have been reported to have neurological disturbances three months after infection by SARS-CoV-2. The mutability of the SARS-COV-2 virus and its potential to directly affect the CNS highlight the urgency of developing technology to diagnose, manage, and treat brain injury in COVID-19 patients. The pathobiology of CNS infection by SARS-CoV-2 and the associated neurological sequelae of this infection remain poorly understood. In this review, we outline the rationale for the use of blood biomarkers (BBs) for diagnosis of brain injury in COVID-19 patients, the research needed to incorporate their use into clinical practice, and the improvements in patient management and outcomes that can result. BBs of brain injury could potentially provide tools for detection of brain injury in COVID-19 patients. Elevations of BBs have been reported in cerebrospinal fluid (CSF) and blood of COVID-19 patients. BB proteins have been analyzed in CSF to detect CNS involvement in patients with infectious diseases, including human immunodeficiency virus and tuberculous meningitis. BBs are approved by the U.S. Food and Drug Administration for diagnosis of mild versus moderate traumatic brain injury and have identified brain injury after stroke, cardiac arrest, hypoxia, and epilepsy. BBs, integrated with other diagnostic tools, could enhance understanding of viral mechanisms of brain injury, predict severity of neurological deficits, guide triage of patients and assignment to appropriate medical pathways, and assess efficacy of therapeutic interventions in COVID-19 patients.

Intracranial Pressure Monitoring in Experimental Traumatic Brain Injury: Implications for Clinical Management.

Traumatic brain injury (TBI) is often associated with long-term disability and chronic neurological sequelae. One common contributor to unfavorable outcomes is secondary brain injury, which is potentially treatable and preventable through appropriate management of patients in the neurosurgical intensive care unit. Intracranial pressure (ICP) is currently the predominant neurological-specific physiological parameter used to direct the care of severe TBI (sTBI) patients. However, recent clinical evidence has called into question the association of ICP monitoring with improved clinical outcome. The detailed cellular and molecular derangements associated with intracranial hypertension (IC-HTN) and their relationship to injury phenotype and neurological outcomes are not completely understood. Various animal models of TBI have been developed, but the clinical applicability of ICP monitoring in the pre-clinical setting has not been well-characterized. Linking basic mechanistic studies in translational TBI models with investigation of ICP monitoring that more faithfully replicates the clinical setting will provide clinical investigators with a more informed understanding of the pathophysiology of IC-HTN, thus facilitating development of improved therapies for sTBI patients.

Chronic Upregulation of Cleaved-Caspase-3 Associated with Chronic Myelin Pathology and Microvascular Reorganization in the Thalamus after Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) is associated with long-term disabilities and devastating chronic neurological complications including problems with cognition, motor function, sensory processing, as well as behavioral deficits and mental health problems such as anxiety, depression, personality change and social unsuitability. Clinical data suggest that disruption of the thalamo-cortical system including anatomical and metabolic changes in the thalamus following TBI might be responsible for some chronic neurological deficits following brain trauma. Detailed mechanisms of these pathological processes are not completely understood. The goal of this study was to evaluate changes in the thalamus following TBI focusing on cleaved-caspase-3, a specific effector of caspase pathway activation and myelin and microvascular pathologies using immuno- and histochemistry at different time points from 24 h to 3 months after controlled cortical impact (CCI) in adult Sprague-Dawley rats. Significant increases in cleaved-caspase-3 immunoreactivity in the thalamus were observed starting one month and persisting for at least three months following experimental TBI. Further, the study demonstrated an association of cleaved-caspase-3 with the demyelination of neuronal processes and tissue degeneration in the gray matter in the thalamus, as reflected in alterations of myelinated fiber integrity (luxol fast blue) and decreases in myelin basic protein (MBP) immunoreactivity. The immunofluorescent counterstaining of cleaved-caspase-3 with endothelial barrier antigen (EBA), a marker of blood-brain barrier, revealed limited direct and indirect associations of cleaved caspase-3 with blood-brain barrier damage. These results demonstrate for the first time a significant chronic upregulation of cleaved-caspase-3 in selected thalamic regions associated with cortical regions directly affected by CCI injury. Further, our study is also the first to report that significant upregulation of cleaved-caspase-3 in selected ipsilateral thalamic regions is associated with microvascular reorganization reflected in the significant increases in the number of microvessels with blood-brain barrier alterations detected by EBA staining. These findings provide new insights into potential mechanisms of TBI cell death involving chronic activation of caspase-3 associated with disrupted cortico-thalamic and thalamo-cortical connectivity. Moreover, this study offers the initial evidence that this upregulation of activated caspase-3, delayed degeneration of myelinated nerve fibers and microvascular reorganization with impaired blood-brain barrier integrity in the thalamus might represent reciprocal pathological processes affecting neuronal networks and brain function at the chronic stages of TBI.

Role of Caspase-3-Mediated Apoptosis in Chronic Caspase-3-Cleaved Tau Accumulation and Blood-Brain Barrier Damage in the Corpus Callosum after Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) may be a significant risk factor for development of neurodegenerative disorders such as chronic traumatic encephalopathy (CTE), post-traumatic epilepsy (PTE), and Alzheimer's (AD) and Parkinson's (PD) diseases. Chronic TBI is associated with several pathological features that are also characteristic of neurodegenerative diseases, including tau pathologies, caspase-3-mediated apoptosis, neuroinflammation, and microvascular alterations. The goal of this study was to evaluate changes following TBI in cleaved-caspase-3 and caspase-3-cleaved tau truncated at Asp421, and their relationships to cellular markers potentially associated with inflammation and blood-brain (BBB) barrier damage. We studied astrocytes (glial fibrillary acidic protein [GFAP]), microglia (ionized calcium-binding adapter molecule 1 [Iba1]), BBB (endothelial barrier antigen [EBA]), and activated microglia/macrophages (cluster of differentiation 68 [CD68]). We employed immunohistochemistry at different time points from 24 h to 3 months after controlled cortical impact (CCI) injury in rats, with particular interest in white matter. The study demonstrated that CCI caused chronic upregulation of cleaved-caspase-3 in the white matter of the corpus callosum. Increases in cleaved-caspase-3 in the corpus callosum were accompanied by accumulation of caspase-3-cleaved tau, with increasing perivascular aggregation 3 months after CCI. Immunofluorescence experiments further showed cellular co-localization of cleaved-caspase-3 with GFAP and CD68 and its adjacent localization with EBA, suggesting involvement of apoptosis and neuroinflammation in mechanisms of delayed BBB and microvascular damage that could contribute to white matter changes. This study also provides the first evidence that evolving upregulation of cleaved-caspase-3 is associated with accumulation of caspase-3-cleaved tau following experimental TBI, thus providing new insights into potential common mechanisms mediated by caspase-3 and underlying chronic TBI pathologies and neurodegenerative diseases.

Prospective clinical biomarkers of caspase-mediated apoptosis associated with neuronal and neurovascular damage following stroke and other severe brain injuries: Implications for chronic neurodegeneration.

Acute brain injuries, including ischemic and hemorrhagic stroke, as well as traumatic brain injury (TBI), are major worldwide health concerns with very limited options for effective diagnosis and treatment. Stroke and TBI pose an increased risk for the development of chronic neurodegenerative diseases, notably chronic traumatic encephalopathy, Alzheimer's disease, and Parkinson's disease. The existence of premorbid neurodegenerative diseases can exacerbate the severity and prognosis of acute brain injuries. Apoptosis involving caspase-3 is one of the most common mechanisms involved in the etiopathology of both acute and chronic neurological and neurodegenerative diseases, suggesting a relationship between these disorders. Over the past two decades, several clinical biomarkers of apoptosis have been identified in cerebrospinal fluid and peripheral blood following ischemic stroke, intracerebral and subarachnoid hemorrhage, and TBI. These biomarkers include selected caspases, notably caspase-3 and its specific cleavage products such as caspase-cleaved cytokeratin-18, caspase-cleaved tau, and a caspase-specific 120 kDa αII-spectrin breakdown product. The levels of these biomarkers might be a valuable tool for the identification of pathological pathways such as apoptosis and inflammation involved in injury progression, assessment of injury severity, and prediction of clinical outcomes. This review focuses on clinical studies involving biomarkers of caspase-3-mediated pathways, following stroke and TBI. The review further examines their prospective diagnostic utility, as well as clinical utility for improved personalized treatment of stroke and TBI patients and the development of prophylactic treatment chronic neurodegenerative disease.

Neuroproteomics and Systems Biology Approach to Identify Temporal Biomarker Changes Post Experimental Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) represents a critical health problem of which diagnosis, management, and treatment remain challenging. TBI is a contributing factor in approximately one-third of all injury-related deaths in the United States. The Centers for Disease Control and Prevention estimate that 1.7 million people suffer a TBI in the United States annually. Efforts continue to focus on elucidating the complex molecular mechanisms underlying TBI pathophysiology and defining sensitive and specific biomarkers that can aid in improving patient management and care. Recently, the area of neuroproteomics-systems biology is proving to be a prominent tool in biomarker discovery for central nervous system injury and other neurological diseases. In this work, we employed the controlled cortical impact (CCI) model of experimental TBI in rat model to assess the temporal-global proteome changes after acute (1 day) and for the first time, subacute (7 days), post-injury time frame using the established cation-anion exchange chromatography-1D SDS gel electrophoresis LC-MS/MS platform for protein separation combined with discrete systems biology analyses to identify temporal biomarker changes related to this rat TBI model. Rather than focusing on any one individual molecular entity, we used in silico systems biology approach to understand the global dynamics that govern proteins that are differentially altered post-injury. In addition, gene ontology analysis of the proteomic data was conducted in order to categorize the proteins by molecular function, biological process, and cellular localization. Results show alterations in several proteins related to inflammatory responses and oxidative stress in both acute (1 day) and subacute (7 days) periods post-TBI. Moreover, results suggest a differential upregulation of neuroprotective proteins at 7 days post-CCI involved in cellular functions such as neurite growth, regeneration, and axonal guidance. Our study is among the first to assess temporal neuroproteome changes in the CCI model. Data presented here unveil potential neural biomarkers and therapeutic targets that could be used for diagnosis, for treatment and, most importantly, for temporal prognostic assessment following brain injury. Of interest, this work relies on in silico bioinformatics approach to draw its conclusion; further work is conducted for functional studies to validate and confirm the omics data obtained.

Animal Models of Posttraumatic Seizures and Epilepsy.

Posttraumatic epilepsy (PTE) is one of the most common and devastating complications of traumatic brain injury (TBI). Currently, the etiopathology and mechanisms of PTE are poorly understood and as a result, there is no effective treatment or means to prevent it. Antiepileptic drugs remain common preventive strategies in the management of TBI to control acute posttraumatic seizures and to prevent the development of PTE, although their efficacy in the latter case is disputed. Different strategies of PTE prophylaxis have been showing promise in preclinical models, but their translation to the clinic still remains elusive due in part to the variability of these models and the fact they do not recapitulate all complex pathologies associated with human TBI. TBI is a multifaceted disorder reflected in several potentially epileptogenic alterations in the brain, including mechanical neuronal and vascular damage, parenchymal and subarachnoid hemorrhage, subsequent toxicity caused by iron-rich hemoglobin breakdown products, and energy disruption resulting in secondary injuries, including excitotoxicity, gliosis, and neuroinflammation, often coexisting to a different degree. Several in vivo models have been developed to reproduce the acute TBI cascade of events, to reflect its anatomical pathologies, and to replicate neurological deficits. Although acute and chronic recurrent posttraumatic seizures are well-recognized phenomena in these models, there is only a limited number of studies focused on PTE. The most used mechanical TBI models with documented electroencephalographic and behavioral seizures with remote epileptogenesis include fluid percussion, controlled cortical impact, and weight-drop. This chapter describes the most popular models of PTE-induced TBI models, focusing on the controlled cortical impact and the fluid percussion injury models, the methods of behavioral and electroencephalogram seizure assessments, and other approaches to detect epileptogenic properties, and discusses their potential application for translational research.

Biomarkers for acute diagnosis and management of stroke in neurointensive care units.

The effectiveness of current management of critically ill stroke patients depends on rapid assessment of the type of stroke, ischemic or hemorrhagic, and on a patient's general clinical status. Thrombolytic therapy with recombinant tissue plasminogen activator (r-tPA) is the only effective treatment for ischemic stroke approved by the Food and Drug Administration (FDA), whereas no treatment has been shown to be effective for hemorrhagic stroke. Furthermore, a narrow therapeutic window and fear of precipitating intracranial hemorrhage by administering r-tPA cause many clinicians to avoid using this treatment. Thus, rapid and objective assessments of stroke type at admission would increase the number of patients with ischemic stroke receiving r-tPA treatment and thereby, improve outcome for many additional stroke patients. Considerable literature suggests that brain-specific protein biomarkers of glial [i.e. S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP)] and neuronal cells [e.g., ubiquitin C-terminal hydrolase-L1 (UCH-L1), neuron-specific enolase (NSE), αII-spectrin breakdown products SBDP120, SBDP145, and SBDP150, myelin basic protein (MBP), neurofilament light chain (NF-L), tau protein, visinin-like protein-1 (VLP 1), NR2 peptide] injury that could be detected in the cerebrospinal fluid (CSF) and peripheral blood might provide valuable and timely diagnostic information for stroke necessary to make prompt management and decisions, especially when the time of stroke onset cannot be determined. This information could include injury severity, prognosis of short-term and long-term outcomes, and discrimination of ischemic or hemorrhagic stroke. This chapter reviews the current status of the development of biomarker-based diagnosis of stroke and its potential application to improve stroke care.

Finding effective biomarkers for pediatric traumatic brain injury.

As traumatic brain injury (TBI) continues to affect children and young adults worldwide, research on reliable biomarkers grows as a possible aid in determining the severity of injury. However, many studies have revealed that diverse biomarkers such as S100B and myelin basic protein (MBP) have many limitations, such as their elevated normative concentrations in young children. Therefore, the results of these studies have yet to be translated to clinical applications. However, despite the setbacks of research into S100B and MBP, investigators continue to research viable biomarkers, notably glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase L1 (UCH-L1), as possible aids in medical decision making. Studies have revealed that GFAP and UCH-L1 actually are better predictors of injury progression than the before-mentioned biomarkers S100B and MBP. In addition, UCH-L1 has demonstrated an ability to detect injury while CT is negative, suggesting an ability to detect acute intracranial lesions. Here, we evaluate research testing levels of GFAP and UCH-L1 on children diagnosed with TBI and compare our results to those of other tested biomarkers. In a recent study done by Hayes et al., GFAP and UCH-L1 demonstrated the potential to recognize children with the possibility of poor outcome, allowing for more specialized treatments with clinical and laboratory applications. Although studies on GFAP and UCH-L1 have for the most part warranted positive results, further studies will be needed to confirm their role as reliable markers for pediatric TBI.

Delayed increases in microvascular pathology after experimental traumatic brain injury are associated with prolonged inflammation, blood-brain barrier disruption, and progressive white matter damage.

Traumatic brain injury (TBI) is a significant risk factor for chronic traumatic encephalopathy (CTE), Alzheimer's disease (AD), and Parkinson's disease (PD). Cerebral microbleeds, focal inflammation, and white matter damage are associated with many neurological and neurodegenerative disorders including CTE, AD, PD, vascular dementia, stroke, and TBI. This study evaluates microvascular abnormalities observed at acute and chronic stages following TBI in rats, and examines pathological processes associated with these abnormalities. TBI in adult rats was induced by controlled cortical impact (CCI) of two magnitudes. Brain pathology was assessed in white matter of the corpus callosum for 24 h to 3 months following injury using immunohistochemistry (IHC). TBI resulted in focal microbleeds that were related to the magnitude of injury. At the lower magnitude of injury, microbleeds gradually increased over the 3 month duration of the study. IHC revealed TBI-induced focal abnormalities including blood-brain barrier (BBB) damage (IgG), endothelial damage (intercellular adhesion molecule 1 [ICAM-1]), activation of reactive microglia (ionized calcium binding adaptor molecule 1 [Iba1]), gliosis (glial fibrillary acidic protein [GFAP]) and macrophage-mediated inflammation (cluster of differentiation 68 [CD68]), all showing different temporal profiles. At chronic stages (up to 3 months), apparent myelin loss (Luxol fast blue) and scattered deposition of microbleeds were observed. Microbleeds were surrounded by glial scars and co-localized with CD68 and IgG puncta stainings, suggesting that localized BBB breakdown and inflammation were associated with vascular damage. Our results indicate that evolving white matter degeneration following experimental TBI is associated with significantly delayed microvascular damage and focal microbleeds that are temporally and regionally associated with development of punctate BBB breakdown and progressive inflammatory responses. Increased understanding of mechanisms underlying delayed microvascular damage following TBI could provide novel insights into chronic pathological responses to TBI and potential common mechanisms underlying TBI and neurodegenerative diseases.

Cerebrospinal fluid protein biomarker panel for assessment of neurotoxicity induced by kainic acid in rats.

Glutamate excitotoxicity plays a key role in the etiology of a variety of neurological, psychiatric, and neurodegenerative disorders. The goal of this study was to investigate spatiotemporal distribution in the brain and cerebrospinal fluid (CSF) concentrations of ubiquitin C-terminal hydrolase-1 (UCH-L1), glial fibrillary acidic protein (GFAP), αII-spectrin breakdown products (SBDP150, SBDP145, and SBDP120), and their relationship to neuropathology in an animal model of kainic acid (KA) excitotoxicity. Triple fluorescent labeling and Fluoro-Jade C staining revealed a reactive gliosis in brain and specific localization of degenerating neurons in hippocampus and entorhinal cortex of KA-treated rats. Immunohistochemistry showed upregulation of GFAP expression in hippocampus and cortex beginning 24h post KA injection and peaking at 48h. At these time points concurrent with extensive neurodegeneration all SBDPs were observed throughout the brain. At 24h post KA injection, a loss of structural integrity was observed in cellular distribution of UCH-L1 that correlated with an increase in immunopositive material in the extracellular matrix. CSF levels of UCH-L1, GFAP, and SBDPs were significantly increased in KA-treated animals compared with controls. The temporal increase in CSF biomarkers correlated with brain tissue distribution and neurodegeneration. This study provided evidence supporting the use of CSF levels of glial and neuronal protein biomarkers to assess neurotoxic damage in preclinical animal models that could prove potentially translational to the clinic. The molecular nature of these biomarkers can provide critical information on the underlying mechanisms of neurotoxicity that might facilitate the development of novel drugs and allow physicians to monitor drug safety.

Hypokalemic nephropathy is associated with impaired angiogenesis.

Hypokalemic nephropathy is associated with alterations in intrarenal vasoactive substances, leading to vasoconstriction, salt-sensitivity, and progression of interstitial fibrosis. In this study, we investigated whether hypokalemic nephropathy might also involve impaired renal angiogenesis. Sprague-Dawley rats that were fed low-potassium diets developed peritubular capillary loss that began in the inner stripe of the outer medulla (week 2) and progressed to the outer stripe of the outer medulla (week 4) and cortex (week 12). These changes were associated with increased macrophage infiltration, increased expression of both monocyte chemoattractant protein-1 and TNF-alpha, and a loss of vascular endothelial growth factor and endothelial nitric oxide synthase. Renal thiobarbituric acid-reactive substances, markers of oxidative stress, were increased late in disease. In conclusion, hypokalemic nephropathy is associated with impaired renal angiogenesis, evidenced by progressive capillary loss, reduced endothelial cell proliferation, and loss of VEGF expression.

IL-10 suppresses chemokines, inflammation, and fibrosis in a model of chronic renal disease.

IL-10 is a pluripotent cytokine that plays a pivotal role in the regulation of immune and inflammatory responses. Whereas short-term administration of IL-10 has shown benefit in acute glomerulonephritis, no studies have addressed the potential benefits of IL-10 in chronic renal disease. Chronically elevated blood levels of IL-10 in rats were achieved by administration of a recombinant adeno-associated virus serotype 1 IL-10 (rAAV1-IL-10) vector. Control rats were given a similar dose of rAAV1-GFP. Four weeks after injection, IL-10 levels in serum were measured by ELISA, and chronic renal disease was induced by a 5/6 nephrectomy (n = 6 in each group). Eight weeks later, rats were killed and renal tissue was obtained for RNA, protein, and immunohistochemical analysis. Serum levels of IL-10 were 12-fold greater in the rAAV1-IL-10 group by 4 wk after rAAV1-IL-10 administration (345 +/- 169 versus 28 +/- 15 pg/ml; P = 0.001), and levels were maintained throughout the experiment. rAAV1-IL-10 treatment resulted in less proteinuria (P < 0.05), lower serum creatinine (P < 0.05), and higher creatinine clearances (P < 0.01) compared with rAAV1-GFP-treated rats. Renal interstitial infiltration was significantly attenuated by rAAV1-IL-10 administration as assessed by numbers of CD4+, CD8+, monocyte-macrophages (ED-1+) and dendritic (OX-62+) cells (P < 0.05), and this correlated with reductions in the renal expression of monocyte (renal monocyte chemoattractant protein-1 mRNA and protein) and T cell (RANTES mRNA) chemokines. rAAV1-IL-10 administration decreased mRNA levels of IFN-gamma and IL-2 in the kidney. The reduction in inflammatory cells was associated with a significant reduction in glomerulosclerosis and interstitial fibrosis. It is concluded that IL-10 blocks inflammation and improves renal function in this model of chronic renal disease. The feasibility of long-term overexpression of a gene using the AAV serotype 1 vector system in a model of renal disease is also demonstrated.

Interleukin 10 attenuates neointimal proliferation and inflammation in aortic allografts by a heme oxygenase-dependent pathway.

Interleukin 10 (IL-10) is a pleiotropic cytokine with well known antiinflammatory, immunosuppressive, and immunostimulatory properties. Chronic allograft rejection, characterized by vascular neointimal proliferation, is a major cause of organ transplant loss, particularly in heart and kidney transplant recipients. In a Dark Agouti to Lewis rat model of aortic transplantation, we evaluated the effects of a single intramuscular injection of a recombinant adeno-associated viral vector (serotype 1) encoding IL-10 (rAAV1-IL-10) on neointimal proliferation and inflammation. rAAV1-IL-10 treatment resulted in a significant reduction of neointimal proliferation and graft infiltration with macrophages and T and B lymphocytes. The mechanism underlying the protective effects of IL-10 in aortic allografts involved heme oxygenase 1 (HO-1) because inhibition of HO activity reversed not only neointimal proliferation but also inflammatory cell infiltration. Our results indicate that IL-10 attenuates neointimal proliferation and inflammatory infiltration and strongly imply that HO-1 is an important intermediary through which IL-10 regulates the inflammatory responses associated with chronic vascular rejection.

Efficient transduction of vascular endothelial cells with recombinant adeno-associated virus serotype 1 and 5 vectors.

Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human alpha1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding beta-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p < 0.001) at 7 days posttransduction. Interestingly, expression was increased in cells transduced with rAAV5 to levels surpassing rAAV1 by day 14 and 21. Transduction with rAAV1 was completely inhibited by removal of sialic acid with sialidase, while heparin had no effect. These studies are the first demonstration that sialic acid residues are required for rAAV1 transduction in endothelial cells. Transduction of rat aortic segments ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies.

Gene delivery in renal tubular epithelial cells using recombinant adeno-associated viral vectors.

Gene therapy has the potential to provide a therapeutic strategy for numerous renal diseases such as diabetic nephropathy, chronic rejection, Alport syndrome, polycystic kidney disease, and inherited tubular disorders. In previous studies using cationic liposomes or adenoviral or retroviral vectors to deliver genes into the kidney, transgene expression has been transient and often associated with adverse host immune responses, particularly with the use of adenoviral vectors. The unique properties of recombinant adeno-associated viral (rAAV) vectors permit long-term stable transgene expression with a relatively low host immune response. The purpose of the present study was to evaluate gene expression in the rat kidney after intrarenal arterial infusion of a rAAV (serotype 2) vector encoding green fluorescence protein (GFP) induced by a cytomegalovirus-chicken beta-actin hybrid promoter. The left kidney of experimental animals was treated with either saline or transduced with rAAV2-GFP (0.125 ml/100 g body wt, 1 x 10(10)/ml infectious units) through the renal artery. A time-dependent expression of GFP was observed in all kidneys injected with rAAV2-GFP, with maximal expression observed at 6 wk posttransduction. The expression of GFP was restricted to cells in the S(3) segment of the proximal tubule and intercalated cells in the collecting duct, the latter identified by co-localization with H(+)-ATPase. No transduction was observed in the glomeruli or the intrarenal vasculature. These studies demonstrate successful transgene expression in tubular epithelial cells, specifically in the S(3) segment of the proximal tubule and intercalated cells, after intrarenal administration of a rAAV vector and provide the impetus for further studies to exploit its use as a tool for gene therapy in the kidney.