New Functional Impairment After Hospital Discharge by Traumatic Brain Injury Mechanism in Younger Than 3 Years Old Admitted to the PICU in a Single Center Retrospective Study.

OBJECTIVES: Children who suffer traumatic brain injury (TBI) are at high risk of morbidity and mortality. We hypothesized that in patients with TBI, the abusive head trauma (AHT) mechanism vs. accidental TBI (aTBI) would be associated with higher frequency of new functional impairment between baseline and later follow-up. DESIGN: Retrospective single center cohort study. SETTING AND PATIENTS: Children younger than 3 years old admitted with TBI to the PICU at a level 1 trauma center between 2014 and 2019. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Patient characteristics, TBI mechanism, and Functional Status Scale (FSS) scores at baseline, hospital discharge, short-term (median, 10 mo [interquartile range 3-12 mo]), and long-term (median, 4 yr [3-6 yr]) postdischarge were abstracted from the electronic health record. New impairment was defined as an increase in FSS greater than 1 from baseline. Patients who died were assigned the highest score (30). Multivariable logistic regression was performed to determine the association between TBI mechanism with new impairment. Over 6 years, there were 460 TBI children (170 AHT, 290 aTBI), of which 13 with AHT and four with aTBI died. Frequency of new impairment by follow-up interval, in AHT vs. aTBI patients, were as follows: hospital discharge (42/157 [27%] vs. 27/286 [9%]; p < 0.001), short-term (42/153 [27%] vs. 26/259 [10%]; p < 0.001), and long-term (32/114 [28%] vs. 18/178 [10%]; p < 0.001). Sensory, communication, and motor domains were worse in AHT patients at the short- and long-term timepoint. On multivariable analysis, AHT mechanism was associated with greater odds (odds ratio [95% CI]) of poor outcome (death and new impairment) at hospital discharge (4.4 [2.2-8.9]), short-term (2.7 [1.5-4.9]), and long-term timepoints (2.4 [1.2-4.8]; p < 0.05). CONCLUSIONS: In patients younger than 3 years old admitted to the PICU after TBI, the AHT mechanism-vs. aTBI-is associated with greater odds of poor outcome in the follow-up period through to ~5 years postdischarge. New impairment occurred in multiple domains and only AHT patients further declined in FSS over time.

Multiorgan Dysfunction Syndrome in Abusive and Accidental Pediatric Traumatic Brain Injury.

BACKGROUND: Abusive head trauma (AHT) is a mechanism of pediatric traumatic brain injury (TBI) with high morbidity and mortality. Multiorgan dysfunction syndrome (MODS), defined as organ dysfunction in two or more organ systems, is also associated with morbidity and mortality in critically ill children. Our objective was to compare the frequency of MODS and evaluate its association with outcome between AHT and accidental TBI (aTBI). METHODS: This was a single center, retrospective cohort study including children under 3 years old admitted to the pediatric intensive care unit with nonpenetrating TBI between 2014 and 2021. Presence or absence of MODS on days 1, 3, and 7 using the Pediatric Logistic Organ Dysfunction-2 score and new impairment status (Functional Status Scale score change > 1 compared with preinjury) at hospital discharge (HD), short-term timepoint, and long-term timepoint were abstracted from the electronic health record. Multiple logistic regression was performed to examine the association between MODS and TBI mechanism with new impairment status. RESULTS: Among 576 children, 215 (37%) had AHT and 361 (63%) had aTBI. More children with AHT had MODS on days 1 (34% vs. 23%, p = 0.003), 3 (28% vs. 6%, p < 0.001), and 7 (17% vs. 3%, p < 0.001) compared with those with aTBI. The most common organ failures were cardiovascular ([AHT] 66% vs. [aTBI] 66%, p = 0.997), neurologic (33% vs. 16%, p < 0.001), and respiratory (34% vs. 15%, p < 0.001). MODS was associated with new impairment in multivariable logistic regression at HD (odds ratio 19.1 [95% confidence interval 9.8-38.6, p < 0.001]), short-term discharge (7.4 [3.7-15.2, p < 0.001]), and long-term discharge (4.3 [2.0-9.4, p < 0.001])]. AHT was also associated with new impairment at HD (3.4 [1.6-7.3, p = 0.001]), short-term discharge (2.5 [1.3-4.7, p = 0.005]), and long-term discharge (2.1 [1.1-4.1, p = 0.036]). CONCLUSIONS: Abusive head trauma as a mechanism was associated with MODS following TBI. Both AHT mechanism and MODS were associated with new impairment at all time points.

Age-related changes in thromboelastography profiles in injured children.

BACKGROUND: The role of age in mediating coagulation characteristics in injured children is not well defined. We hypothesize thromboelastography (TEG) profiles are unique across pediatric age groups. METHODS: Consecutive trauma patients younger than 18 years from a Level I pediatric trauma center database from 2016 to 2020 with TEG obtained on arrival to the trauma bay were identified. Children were categorized by age according to the National Institute of Child Health and Human Development categories (infant, ≤1 year; toddler, 1-2 years; early childhood, 3-5 years; older childhood, 6-11 years; adolescent, 12-17 years). Thromboelastography values were compared across age groups using Kruskal-Wallis and Dunn's tests. Analysis of covariance was performed controlling for sex, Injury Severity Score (ISS), arrival Glasgow Coma Scale (GCS) score, shock, and mechanism of injury. RESULTS: In total, 726 subjects were identified; 69% male, median (interquartile range [IQR]) ISS = 12 (5-25), and 83% had a blunt mechanism. On univariate analysis, there were significant differences in TEG α-angle ( p < 0.001), MA ( p = 0.004), and fibrinolysis 30 minutes after MA (LY30) ( p = 0.01) between groups. In post hoc tests, the infant group had significantly greater α-angle (median, 77; IQR, 71-79) and MA (median, 64; IQR, 59-70) compared with other groups, while the adolescent group had significantly lower α-angle (median, 71; IQR, 67-74), MA (median, 60; IQR, 56-64), and LY30 (median, 0.8; IQR, 0.2-1.9) compared with other groups. There were no significant differences between toddler, early childhood, and middle childhood groups. On multivariate analysis, the relationship between age group and TEG values (α-angle, MA, and LY30) persisted after controlling for sex, ISS, GCS, shock, and mechanism of injury. CONCLUSION: Age-associated differences in TEG profiles across pediatric age groups exist. Further pediatric-specific research is required to assess whether the unique profiles at extremes of childhood translate to differential clinical outcomes or responses to therapies in injured children. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level IV.

Association of Prehospital Transfusion With Mortality in Pediatric Trauma.

Importance: Optimal hemostatic resuscitation in pediatric trauma is not well defined. Objective: To assess the association of prehospital blood transfusion (PHT) with outcomes in injured children. Design, Setting, and Participants: This retrospective cohort study of the Pennsylvania Trauma Systems Foundation database included children aged 0 to 17 years old who received a PHT or emergency department blood transfusion (EDT) from January 2009 and December 2019. Interfacility transfers and isolated burn mechanism were excluded. Analysis took place between November 2022 and January 2023. Exposure: Receipt of a blood product transfusion in the prehospital setting compared with the emergency department. Main Outcomes and Measures: The primary outcome was 24-hour mortality. A 3:1 propensity score match was developed balancing for age, injury mechanism, shock index, and prehospital Glasgow Comma Scale score. A mixed-effects logistic regression was performed in the matched cohort further accounting for patient sex, Injury Severity Score, insurance status, and potential center-level heterogeneity. Secondary outcomes included in-hospital mortality and complications. Results: Of 559 children included, 70 (13%) received prehospital transfusions. In the unmatched cohort, the PHT and EDT groups had comparable age (median [IQR], 47 [9-16] vs 14 [9-17] years), sex (46 [66%] vs 337 [69%] were male), and insurance status (42 [60%] vs 245 [50%]). The PHT group had higher rates of shock (39 [55%] vs 204 [42%]) and blunt trauma mechanism (57 [81%] vs 277 [57%]) and lower median (IQR) Injury Severity Score (14 [5-29] vs 25 [16-36]). Propensity matching resulted in a weighted cohort of 207 children, including 68 of 70 recipients of PHT, and produced well-balanced groups. Both 24-hour (11 [16%] vs 38 [27%]) and in-hospital mortality (14 [21%] vs 44 [32%]) were lower in the PHT cohort compared with the EDT cohort, respectively; there was no difference in in-hospital complications. Mixed-effects logistic regression in the postmatched group adjusting for the confounders listed above found PHT was associated with a significant reduction in 24-hour (adjusted odds ratio, 0.46; 95% CI, 0.23-0.91) and in-hospital mortality (adjusted odds ratio, 0.51; 95% CI, 0.27-0.97) compared with EDT. The number needed to transfuse in the prehospital setting to save 1 child's life was 5 (95% CI, 3-10). Conclusions and Relevance: In this study, prehospital transfusion was associated with lower rates of mortality compared with transfusion on arrival to the emergency department, suggesting bleeding pediatric patients may benefit from early hemostatic resuscitation. Further prospective studies are warranted. Although the logistics of prehospital blood product programs are complex, strategies to shift hemostatic resuscitation toward the immediate postinjury period should be pursued.

Association of Thromboelastography with Progression of Hemorrhagic Injury in Children with Traumatic Brain Injury.

INTRODUCTION: Progression of hemorrhagic injury (PHI) in children with traumatic brain injury portends poor outcomes. The association between thromboelastography (TEG), functional coagulation assays, and PHI is not well characterized in children. METHODS: This was a retrospective cohort study of children presenting with PHI at a pediatric level I academic trauma center from 2015 to 2020. Inclusion criteria were as follows: age less than 18 years, intracranial hemorrhage on admission head computed tomography scan, and admission rapid TEG assay and conventional coagulation tests. PHI was defined by the following radiographic criteria: any expansion of or new intracranial hemorrhage on subsequent head computed tomography scan. Rapid TEG values included Activated Clotting Time (ACT), alpha angle, maximum amplitude, and lysis at 30 min. Wilcoxon rank-sum test was used to assess baseline differences between groups with PHI and without PHI, including laboratory assays. Univariate analysis was performed to examine the association between variables of interest and PHI. Patients were dichotomized on the basis of this cut point to generate a "low ACT" group and a "high ACT" group. These variables were included in a multivariable logistic regression model to determine independent association with traumatic brain injury progression. RESULTS: In total, 219 patients met criteria for analysis. In this cohort, the median (interquartile range [IQR]) age = 6 (2-12) years, median (IQR) Injury Severity Score = 21 (11-27), 68% were boys, and 69% sustained blunt injury. The rate of PHI was 25% (54). Median (IQR) time to PHI was 1 (0-4) days. Children with PHI had a higher Injury Severity Score (p < 0.001), lower Glasgow Coma Scale (p < 0.001), greater incidence of shock (p = 0.04), and lower admission hemoglobin (p = 0.02) compared with those without PHI. Children with PHI had a higher International Normalized Ratio (INR) and longer TEG-ACT; other TEG values (alpha angle, maximum amplitude, and lysis at 30 min) were not associated with PHI. In the logistic regression model accounting for other covariates associated with PHI, elevated ACT remained an independent predictor of progression (odds ratio = 2.25, 95% confidence interval 1.09-4.66; p = 0.03; area under the receiver operating characteristic curve = 0.76). After adjusting for confounders, INR fell out of the model and was not an independent predictor of progression (odds ratio = 1.32, 95% confidence interval 0.60-2.93; p = 0.49). CONCLUSIONS: Although INR was elevated in children with PHI and has been associated with poor clinical outcomes, only admission TEG-ACT was independently associated with PHI. Further study is warranted to determine whether TEG-ACT reflects an actionable therapeutic target.

Recognizing life-threatening bleeding in pediatric trauma: A standard for when to activate massive transfusion protocol.

BACKGROUND: Traumatic hemorrhage is the most common cause of preventable death in civilian and military trauma. Early identification of pediatric life-threatening hemorrhage is challenging. There is no accepted clinical critical administration threshold (CAT) in children for activating massive transfusion protocols. METHODS: Children 0 to 17 years old who received any transfusion in the first 24 hours after injury between 2010 and 2019 were included. The type, volume, and time of administration for each product were recorded. The greatest volume of weight-adjusted products transfused within 1 hour was calculated. The cut point for the number of products that maximized sensitivity and specificity to predict in-hospital mortality, need for urgent surgery, and second life-threatening bleeding episode was determined using Youden's index. A binary variable (CAT+) was generated using this threshold for inclusion in a multivariable logistic regression model. RESULTS: In total, 287 patients were included. The median (interquartile range) age was 6 (2-14) years, 60% were males, 83% sustained blunt trauma, and the median (interquartile range) Injury Severity Score was 26 (17-35). The optimal cutoff to define CAT+ was >20 mL/kg of product; this optimized test characteristics for mortality (sensitivity, 70%; specificity, 77%), need for urgent hemorrhage control procedure (sensitivity, 65%; specificity, 74%). and second bleeding episode (sensitivity, 77%; specificity, 74%). There were 93 children (32%) who were CAT+. On multivariate regression, being CAT+ was associated with 3.4 increased odds of mortality (95% confidence interval, 1.67-6.89; p = 0.001) after controlling for age, hypotension, Injury Severity Score, and Glasgow Coma Scale. For every unit of product administered, there was a 10% increased risk of mortality (odds ratio, 1.1; p < 0.001). CONCLUSION: Transfusion of more than 20 mL/kg of any blood product within an hour should be used as a threshold for activating massive transfusion protocols in children. Children who meet this CAT are at high risk of mortality and need for interventions; this population may benefit from targeted, timely, and aggressive hemostatic resuscitation. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level III.

Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS-NO-nitrite signaling.

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS(-/-) mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate--a precursor for enteral generation of nitrite and nitric oxide--and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate-nitrite-NO signaling.

A critical role for TLR4 induction of autophagy in the regulation of enterocyte migration and the pathogenesis of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) develops in response to elevated TLR4 signaling in the newborn intestinal epithelium and is characterized by TLR4-mediated inhibition of enterocyte migration and reduced mucosal healing. The downstream processes by which TLR4 impairs mucosal healing remain incompletely understood. In other systems, TLR4 induces autophagy, an adaptive response to cellular stress. We now hypothesize that TLR4 induces autophagy in enterocytes and that TLR4-induced autophagy plays a critical role in NEC development. Using mice selectively lacking TLR4 in enterocytes (TLR4(ΔIEC)) and in TLR4-deficient cultured enterocytes, we now show that TLR4 activation induces autophagy in enterocytes. Immature mouse and human intestine showed increased expression of autophagy genes compared with full-term controls, and NEC development in both mouse and human was associated with increased enterocyte autophagy. Importantly, using mice in which we selectively deleted the autophagy gene ATG7 from the intestinal epithelium (ATG7(ΔIEC)), the induction of autophagy was determined to be required for and not merely a consequence of NEC, because ATG7(ΔIEC) mice were protected from NEC development. In defining the mechanisms involved, TLR4-induced autophagy led to impaired enterocyte migration both in vitro and in vivo, which in cultured enterocytes required the induction of RhoA-mediated stress fibers. These findings depart from current dogma in the field by identifying a unique effect of TLR4-induced autophagy within the intestinal epithelium in the pathogenesis of NEC and identify that the negative consequences of autophagy on enterocyte migration play an essential role in its development.

Intestinal stem cells and their roles during mucosal injury and repair.

The ability of the host to respond to intestinal injury requires the regeneration of native tissue through a highly orchestrated response from the intestinal stem cells, a population of cells located within the intestinal crypts that have the capability to repopulate the entire villous. The field of intestinal stem cell biology is thus of great interest to surgeons and non-surgeons alike, given its relevance to diseases of intestinal injury and inflammation such as inflammatory bowel disease, trauma, and necrotizing enterocolitis. The field of intestinal stem cell research has been advanced recently by the identification of the putative marker, Lgr5, which has allowed for the isolation and further characterization of the intestinal stem cell. Under the control of the WNT signaling pathway, Lgr5 marks the rapidly dividing cells of the intestinal crypt, and identifies a population of cells that is capable of regenerating the entire villous. We now review the identification of Lgr5 as an intestinal stem cell marker, identify controversies in the intestinal stem cell field, and highlight the response of the intestinal stem cell to injury within the intestinal mucosa that may occur clinically.

Nucleotide-binding oligomerization domain-2 inhibits toll-like receptor-4 signaling in the intestinal epithelium.

BACKGROUND & AIMS: Factors that regulate enterocyte apoptosis in necrotizing enterocolitis (NEC) remain incompletely understood, although Toll-like receptor-4 (TLR4) signaling in enterocytes plays a major role. Nucleotide-binding oligomerization domain-2 (NOD2) is an immune receptor that regulates other branches of the immune system, although its effects on TLR4 in enterocytes and its role in NEC remain unknown. We now hypothesize that activation of NOD2 in the newborn intestine inhibits TLR4, and that failure of NOD2 signaling leads to NEC through increased TLR4-mediated enterocyte apoptosis. METHODS: The effects of NOD2 on enterocyte TLR4 signaling and intestinal injury and repair were assessed in enterocytes lacking TLR4 or NOD2, in mice with intestinal-specific wild-type or dominant-negative TLR4 or NOD2, and in mice with NEC. A protein array was performed on NOD2-activated enterocytes to identify novel effector molecules involved. RESULTS: TLR4 activation caused apoptosis in newborn but not adult small intestine or colon, and its intestinal expression was influenced by NOD2. NOD2 activation inhibited TLR4 in enterocytes, but not macrophages, and reversed the effects of TLR4 on intestinal mucosal injury and repair. Protection from TLR4-induced enterocyte apoptosis by NOD2 required a novel pathway linking NOD2 with the apoptosis mediator second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI (SMAC-DIABLO), both in vitro and in vivo. Strikingly, activation of NOD2 reduced SMAC-DIABLO expression, attenuated the extent of enterocyte apoptosis, and reduced the severity of NEC. CONCLUSIONS: These findings reveal a novel inhibitory interaction between TLR4 and NOD2 signaling in enterocytes leading to the regulation of enterocyte apoptosis and suggest a therapeutic role for NOD2 in the protection of intestinal diseases such as NEC.

Toll-like receptor-4 inhibits enterocyte proliferation via impaired beta-catenin signaling in necrotizing enterocolitis.

BACKGROUND & AIMS: Necrotizing enterocolitis (NEC), the leading cause of gastrointestinal death from gastrointestinal disease in preterm infants, is characterized by exaggerated TLR4 signaling and decreased enterocyte proliferation through unknown mechanisms. Given the importance of beta-catenin in regulating proliferation of many cell types, we hypothesize that TLR4 impairs enterocyte proliferation in NEC via impaired beta-catenin signaling. METHODS: Enterocyte proliferation was detected in IEC-6 cells or in ileum or colon from wild-type, TLR4-mutant, or TLR4(-/-) mice after induction of NEC or endotoxemia. beta-Catenin signaling was assessed by cell fractionation or immunoconfocal microscopy to detect its nuclear translocation. Activation and inhibition of beta-catenin were achieved via cDNA or small interfering RNA, respectively. TLR4 in the intestinal mucosa was inhibited with adenoviruses expressing dominant-negative TLR4. RESULTS: TLR4 activation significantly impaired enterocyte proliferation in the ileum but not colon in newborn but not adult mice and in IEC-6 enterocytes. beta-Catenin activation reversed these effects in vitro. To determine the mechanisms involved, TLR4 activation phosphorylated the upstream inhibitory kinase GSK3beta, causing beta-catenin degradation. NEC in both mouse and humans was associated with decreased beta-catenin and increased mucosal GSK3beta expression. Strikingly, the inhibition of enterocyte beta-catenin signaling in NEC could be reversed, and enterocyte proliferation restored, through adenoviral-mediated inhibition of TLR4 signaling in the small intestinal mucosa. CONCLUSION: We now report a novel pathway linking TLR4 with inhibition of beta-catenin signaling via GSK3beta activation, leading to reduced enterocyte proliferation in vitro and in vivo. These data provide additional insights into the pathogenesis of diseases of intestinal inflammation such as NEC.

Reciprocal expression and signaling of TLR4 and TLR9 in the pathogenesis and treatment of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a common and often fatal inflammatory disorder affecting preterm infants that develops upon interaction of indigenous bacteria with the premature intestine. We now demonstrate that the developing mouse intestine shows reciprocal patterns of expression of TLR4 and TLR9, the receptor for bacterial DNA (CpG-DNA). Using a novel ultrasound-guided in utero injection system, we administered LPS directly into the stomachs of early and late gestation fetuses to induce TLR4 signaling and demonstrated that TLR4-mediated signaling within the developing intestine follows its expression pattern. Murine and human NEC were associated with increased intestinal TLR4 and decreased TLR9 expression, suggesting that reciprocal TLR4 and TLR9 signaling may occur in the pathogenesis of NEC. Enteral administration of adenovirus expressing mutant TLR4 to neonatal mice reduced the severity of NEC and increased TLR9 expression within the intestine. Activation of TLR9 with CpG-DNA inhibited LPS-mediated TLR4 signaling in enterocytes in a mechanism dependent upon the inhibitory molecule IRAK-M. Strikingly, TLR9 activation with CpG-DNA significantly reduced NEC severity, whereas TLR9-deficient mice exhibited increased NEC severity. Thus, the reciprocal nature of TLR4 and TLR9 signaling within the neonatal intestine plays a role in the development of NEC and provides novel therapeutic approaches to this disease.

No longer an innocent bystander: epithelial toll-like receptor signaling in the development of mucosal inflammation.

Diseases of mucosal inflammation represent important causes of morbidity and mortality, and have led to intense research efforts to understand the factors that lead to their development. It is well accepted that a breakdown of the normally impermeant epithelial barrier of the intestine, the lung, and the kidney is associated with the development of inflammatory disease in these organs, yet significant controversy exists as to how this breakdown actually occurs, and how such a breakdown may lead to inflammation. In this regard, much work has focused upon the role of the epithelium as an "innocent bystander," a target of a leukocyte-mediated inflammatory cascade that leads to its destruction in the mucosal inflammatory process. However, recent evidence from a variety of laboratories indicates that the epithelium is not merely a passive component in the steps that lead to mucosal inflammation, but is a central participant in the process. In addressing this controversy, we and others have determined that epithelial cells express Toll-like receptors (TLRs) of the innate immune system, and that activation of TLRs by endogenous and exogenous ligands may play a central role in determining the balance between a state of "mucosal homeostasis," as is required for optimal organ function, and "mucosal injury," leading to mucosal inflammation and barrier breakdown. In particular, activation of TLRs within intestinal epithelial cells leads to the development of cellular injury and impairment in mucosal repair in the pathogenesis of intestinal inflammation, while activation of TLRs in the lung and kidney may participate in the development of pneumonitis and nephritis respectively. Recent work in support of these concepts is extensively reviewed, while essential areas of further study that are required to determine the significance of epithelial TLR signaling during states of health and disease are outlined.