nature publishing group Basic Science Investigation Articles Hypoxic–ischemic brain damage induces distant inflammatory lung injury in newborn piglets Luis Arruza1, M. Ruth Pazos2, Nagat Mohammed2, Natalia Escribano3, Hector Lafuente4, Martín Santos2, Francisco J. Alvarez-Díaz4 and Jose Martínez-Orgado1,2 BACKGROUND: We aimed to investigate whether neonatal the incidence of lung injury is related to the severity of brain hypoxic–ischemic (HI) brain injury induces inflammatory lung damage and represents an independent factor associated with damage. poor clinical outcome (7). METHODS: Thus, hypoxic (HYP, FiO2 10% for 30 min), isch- Although the etiology of respiratory dysfunction after brain emic (ISC, bilateral carotid flow interruption for 30 min), or HI damage in adults is not completely understood, inflamma- event was performed in 1–2-d-old piglets. Dynamic compli- tion is thought to play a key role, together with other mecha- ance (Cdyn), oxygenation index (OI), and extravascular lung nisms such as neurogenic pulmonary edema, left ventricular water (EVLW) were monitored for 6 h. Then, histologic dam- dysfunction, or infection (8,9). In adult animal models and in age was assessed in brain and lung (lung injury severity score). patients suffering from subarachnoid hemorrhage, brain injury Total protein content (TPC) was determined in broncoalveolar leads to a systemic inflammatory response with upregulation lavage fluid (BALF), and IL-1β concentration was measured in of inflammatory mediators such as TNF-α, intracellular adhe- lung and brain tissues and blood. sion molecule-1, IL-1β, and NF-kB and reactive oxygen species, RESULTS: Piglets without hypoxia or ischemia served as con- microglial activation, and neutrophil accumulation (5,9,10). It trols (SHM). HI-induced brain damage was associated with has been hypothesized that lung injury might be triggered by decreased Cdyn, increased OI and EVLW, and histologic lung the spread of that (such a systemic) response, favored by the damage (interstitial leukocyte infiltration, congestive hyper- disruption of the blood–brain barrier in the context of this emia, and interstitial edema). BALF TPC was increased, sug- inflammatory process (10). Such a cascade of events is not gesting inflammatory damage. In agreement, tissue IL-1β privative for the brain. For instance, bilateral renal ischemia is concentration increased in the brain and lung, in correspon- followed by an increase in serum IL-1, IL-6, and IL-12 and lung dence with increased IL-1β serum concentration. Neither HYP injury in mice (11). Similarly, intestinal ischemia–reperfusion nor ISC alone led to brain or lung damage. injury induces acute lung injury in rats, with cellular infiltra- CONCLUSION: HI brain damage in newborn piglets led to tion, microvascular dysfunction, and interstitial edema (12). inflammatory lung damage, suggesting an additional mecha- Since systemic inflammation and oxidative stress, together nism accounting for the development of lung dysfunction with blood–brain barrier disruption, are events typically tak- after neonatal HI encephalopathy. ing place in NHIE pathophysiology (13,14), it is attractive to hypothesize that the aforementioned mechanisms for distant lung injury after brain damage might be involved in the lung amage of organs other than the brain often complicates dysfunction accompanying newborn HIE. Previous stud- Dneonatal hypoxic–ischemic (HI) encephalopathy (NHIE) ies reported that lung compliance and gas interchange are after perinatal asphyxia (1,2). There is a stepwise increase impaired in newborn piglets after brain HI insults (15,16), in the rate of adverse outcomes as the number of additional pointing to the existence of lung injury in that scenario. organs involved increase in newborns with NHIE (2). Lungs The aim of this work was to demonstrate specifically the are often affected: depending on the definition criteria, 25 to existence of brain HI-induced lung injury and to study the 86% of HIE infants develop signs of respiratory dysfunction underlying mechanisms using a newborn piglet model of HI (1,2). Extracerebral impact of perinatal asphyxia has been uni- brain damage. formly attributed to the redistribution of blood flow and/or the effects of global hypoxia–ischemia (2,3). Observational studies RESULTS from adults indicate that lung injury may develop after local Homeostatic Parameters brain damage such as intracranial hemorrhage, traumatic brain Table 1 shows the homeostatic parameters of the four differ- injury, or ischemic cerebral stroke (4–6). For these conditions, ent groups. No statistically significant differences were found 1Department of Neonatology, Hospital Clínico San Carlos, Madrid, Spain; 2Health Research Institute Puerta de Hierro Majadahonda, Madrid, Spain; 3Department of ­Pathology, Hospital Clínico San Carlos, Madrid, Spain; 4Intensive Care Traslational Research Group, Biocruces Health Research Institute, Bizkaia, Spain. Correspondence: Jose Martínez-Orgado ([email protected]) Received 21 October 2014; accepted 26 January 2015; advance online publication 27 May 2015. doi:10.1038/pr.2015.87 Copyright © 2016 International Pediatric Research Foundation, Inc. Volume 79 | Number 3 | March 2016 Pediatric Research 401 Articles Arruza et al. between groups in cardiac output at all time points. However, in somatic rSO2. Hematocrit remained stable throughout the HI led to a significant drop in mean arterial blood pressure that experiment for all groups. persisted throughout the experimental period, so that half the number of HI piglets required substantial inotropic support Brain Damage (dopamine, mean dose: 13 ± 4 μg/kg/min) to maintain appro- Assessment of brain damage. Continued sedoanalgesia priate mean arterial blood pressure values. Interestingly, such determined that amplitude-integrated EEG (aEEG) ampli- a drop in mean arterial blood pressure was not observed in the tude decreased slightly in SHM animals throughout the hypoxic (HYP) or ischemic (ISC) groups, with only one piglet experiment (Figure 1), although this effect was not associ- from each of those groups receiving inotropic support (dopa- ated with an impairment of background EEG activity and/ mine 7.5 μg/kg/min) by the end of the experiment. No control or the EEG pattern (data not shown). HI led to a dramatic (SHM) piglet needed inotropic support. Whereas no variation decrease in brain activity that did not recover during the fol- in pCO2 was observed in piglets throughout the experiment, a lowing 6 h (Figure 1). Hypoxia and ischemia alone led to a transient but significant decrease in blood pH and in cerebral slight decrease in aEEG amplitude during the insult, but in and somatic rSO2 was observed during the hypoxemic period this case, aEEG regained normal amplitude to the end of the in HI and HYP groups. Brain ischemia alone, as in ISC, led to experiment. Throughout the experiment, aEEG amplitude in a milder but significant drop in cerebral rSO2 with no changes HYP or ISC was statistically significantly higher than that in HI (Figure 1). Analysis of Nissl-stained brain tissue revealed a reduced Table 1. Homeostatic parameters number of normal neurons in HI as compared with SHM with HI HYP ISC a 5-fold increase in the percentage of nonviable neurons in the Parameter SHM (n = 6) (n = 8) (n = 7) (n = 5) frontoparietal cortex and a 10-fold increase in the hippocam- CO (ml/ B 318.4 (29) 356.9 (45) 329.0 (21) 313.1 (40) pus (Figure 2a). Brain histology was similar in HYP and ISC min/100 g) I 340.4 (24) 355.7 (34) 370.0 (40) 330 (45) to SHM. E 350.2 (36) 344.3 (42) 365.7 (28) 350.5 (36) HI-induced neuroinflammation. Cerebral HI induced a MABP B 76.3 (7) 80.8 (3) 79.3 (5) 70.6 (5.8) (mm Hg) local inflammatory response as evidenced by an increase in I 83.5 (6) 56.4 (8)* 82.1 (4) 70.0 (7) IL-1β concentration in brain tissue as compared with SHM E 78.8 (5) 59.6 (3)* 71.7 (4) 70.5 (5) (Figure 3). Brain IL-1β concentration was similar in HYP or pH B 7.34 (0.02) 7.32 (0.02) 7.32 (0.01) 7.36 (0.01) ISC to SHM. I 7.38 (0.02) 7.20 (0.02)* 7.23 (0.03)* 7.33 (0.01) Blood Inflammatory Markers E 7.38 (0.01) 7.32 (0.04) 7.34 (0.05) 7.35 (0.03) HI led to an increase in IL-1β serum levels as compared with pCO (mm Hg) B 41.3 (2.4) 34.9 (2.6) 39.7 (3.3) 39 (1.9) 2 SHM–an increase not observed in HYP or ISC (Figure 3). I 39.3 (2.3) 42.4 (2.2) 38.5 (3.1) 43.0 (1.6) Interestingly, a positive correlation was found between brain 2 E 38.8 (1.1) 42.2 (2.9) 38.7 (2.7) 43.6 (2.5) and serum IL-1β concentrations (R = 0.75; P < 0.05). Base excess B −2.95 (1,2) −3.55 (0.8) −4.1 (0.7) −2.1 (1.5) 30 I −1.58 (1) −12.8 (1.2)* −12.0 (1.6)* −1.9 (0.9)§ E −1.17 (1.2) −4.2 (1.8) −3.6 (0.9) −0.97 (1.8) 25 SpO2 (%) B 96.6 (0.8) 96.2 (1.1) 97.0 (0.9) 98.9 (0.5) I 96.1 (2.2) 83.2 (1.9)* 85.7 (2.1)* 98.3 (3.0) 20 * E 100 (1.1) 96.7 (0.7) 96.0 (1.9) 95.1 (2.1) * V 15 * µ rSO2 (C) (%) B 55.0 (2.7) 52.6 (1.8) 53.0 (2.8) 49.6 (2.4) I 55.1 (2.7) 19.1 (3.3)* 28.0 (1.4)*§ 44.0 (2.2)*§ 10 § § § E 54.1 (2.7) 50.6 (1.3) 53.2 (2.6) 54.6 (2.7) § 5 rSO2 (S) (%) B 55.0 (2.7) 56.2 (5.2) 54.2 (4.8) 52.6 (2.6) I 55.0 (2.7) 24.1 (2.7)* 21.4 (6.1)* 54.2 (2.7) 0 E 51.3 (2.5) 52.6 (5.6) 58.0 (8.0) 51.3 (2.5) B I 1 3 6 Hours after insult Ht (%) B 18.5 (1.9) 20.5 (1.2) 21 (1.2) 19.3 (1.2) E 20.0 (1.8) 20.3 (1.5) 22.0 (1.4) 22.5 (1.4) Figure 1.