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Blood–Brain Barrier Dysfunction After Smoke Inhalation Injury, With and Without Skin Burn

Randolph, Anita C.*; Fukuda, Satoshi*; Ihara, Koji†,‡; Enkhbaatar, Perenlei*,§; Micci, Maria-Adelaide*

doi: 10.1097/SHK.0000000000001196
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ABSTRACT Only a handful of published reports exist today that describe neurological complications following smoke inhalation injury. In this study, we characterize acute pathophysiological changes in the brain of sheep exposed to smoke inhalation, with- and without third-degree skin burn that models the injuries sustained by human victims of fire accidents. Blood–brain barrier integrity and hemorrhage were analyzed throughout the brain using specific histological stains: Hematoxylin & Eosin, Luxol fast blue, Periodic acid–Schiff (PAS), and Martius, Scarlet and Blue (MSB). Our data show that, following smoke inhalation injury, alone and in combination with third-degree skin burn, there was a significant increase in the number of congested and dilated blood vessels in the frontal cortex, basal ganglia, amygdala, hippocampus, pons, cerebellum, and pituitary gland as compared to sham-injured controls. Positive PAS staining confirmed damage to the basement membrane of congested and dilated blood vessels throughout the brain. Severe rupturing of blood vessels, microvascular hemorrhaging and bleeding throughout the brain was also observed in the injured groups. No significant changes in hemodynamics and PaO2 were observed. Our data demonstrate for the first time that acute smoke inhalation alone results in diffuse blood-brain barrier dysfunction and massive bleeding in the brain in the absence of hypoxia and changes in hemodynamics. These findings provide critical information and prompt further mechanistic and interventional studies necessary to develop effective and novel treatments aimed at alleviating CNS dysfunction in patients with smoke and burn injuries.

*Department of Anesthesiology, University of Texas Medical Branch, Galveston, Texas

Department of Plastic and Reconstructive Surgery, Kagoshima City Hospital, Kagoshima

Department of Plastic and Reconstructive Surgery, Tokyo Women's Medical School, Tokyo, Japan

§Shriner's Hospital for Children, Galveston, Texas

Address reprint requests to Anita C. Randolph, PhD, Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX 77555. E-mail: anrandol@utmb.edu

Received 16 March, 2018

Revised 3 April, 2018

Accepted 24 May, 2018

This work was supported by two grants from Shriners of North America (Tampa, Florida), “Special Shared Facility Lung Lymph Laboratory” (Shrine No. 84050, Enkhbaatar, PI), and “Adipose-derived Stem Cell Therapy for Lung Injury After Burn and Smoke Inhalation” (Shrine No. 85100, Enkhbaatar, PI).

The authors report no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the journal's Web site (www.shockjournal.com).

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INTRODUCTION

Smoke inhalation injury

Each year, more than 23,000 smoke inhalation injuries are reported in the United States (1). The shift from the use of natural to synthetic materials and petrochemicals has dramatically increased the complexity of toxic gases inhaled after ignition, thereby increasing the likelihood of inhalation injury after combustion (2). Tragic events such as the terrorist attacks on the World Trade Center and the Pentagon in 2001 and the Kiss nightclub fire in Brazil in 2013 were associated with high incidents of smoke-inhalation injury (3, 4). Specifically, inhalation of toxic compounds present in the dust and smoke during the World Trade Center attack caused 49% of inhalation injuries (3, 4). Also, the World Health Organization (WHO) reported that inhalation of smoke generated from indoor cooking, forest fires, and burning crops leads to more than 1 billion cases of airway and pulmonary inflammation each year (5).

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Burn injury

The American Burn Association (ABA) reports that in 2015 there were 203,422 burn injuries that required some degree of medical attention in the United States (6). While thermal burns account for 42.6% of all burn injuries, injuries caused by fire and flame account for the majority of medically treated burns (6). Between 2% and 30% of burn patients with thermal injuries from fires suffer from combined burn and smoke inhalation injury that greatly increases their morbidity and mortality (7).

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CNS dysfunction following injury

There is limited information available regarding the effects of smoke inhalation with and without third-degree skin burn on the central nervous system (CNS). Despite the fact that advances in clinical care and the development of novel treatments have greatly increased the survival of burn-injury and smoke inhalation injury patients, as of today, very few follow-up studies on pathological alterations of the CNS and associated cognitive dysfunctions have been conducted.

Neurological complications such as a persistent headache, memory loss, and paresthesia (abnormal sensation such as tingling with no physical stimulation) were reported in survivors of the 2013 nightclub fire that suffered smoke inhalation injuries (3). In one clinical report, a progressive decline in neurological functions was reported after a single acute combustible smoke inhalation incident (8). Specifically, complications started within one-month post-injury and included headache, anhedonia (inability to feel pleasure from normally pleasurable activities), impaired concentration, and reduced attention and learning skills. Cognitive impairments such as difficulty in word finding, bradyphrenia (slowness of thought), and reduced short and long-term memory were also reported. Additionally, positron emission tomography (PET) showed decreased metabolic activity in the entire brain three years postinjury, and follow-up PET scan, 14 years postinjury, revealed areas of continued deterioration in the corpus callosum, orbital frontal lobes, globus pallidus, putamen, and thalamus. Most important, although there were areas of increased glucose metabolism, none of the areas returned to normal control values over the time of observation. These reports suggest that long-term neurological and cognitive impairments occur as a result of smoke inhalation and prompts the need for more extensive characterizations of CNS pathological changes following smoke inhalation with and without third-degree skin burn.

In this study, we used an established and clinically relevant ovine model of smoke inhalation and third-degree skin burn that reproduces injuries suffered by victims of fire such as firefighters, factory, and construction workers (2) to characterize pathological changes in the brain with a focus on blood–brain barrier damages and related structural changes.

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MATERIALS AND METHODS

Animal compliance and use

This study was completed in compliance with the National Institutes of Health Office of Animal Welfare and approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch.

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Surgical catheterization of sheep in preparation for injury

Twenty-three female Merino sheep between 30 and 40 kg were surgically prepped with multiple vascular catheters for hemodynamic monitoring during the experimental period (9). In preparation for surgery, female sheep were deeply anesthetized using ketamine (KetaVed, Phoenix Scientific, St. Joseph, Mo), both intramuscular (IM) and intravenous (IV), followed by 5% isoflurane (IsoSol, VEDCO, St. Joseph, Mo) via an endotracheal tube. To avoid discomfort, a long-acting analgesia (0.005–0.01 mg/kg of buprenorphine slow release (SR)) (Buprenorphine SR, SR Veterinary Technologies, Windsor, Colo) was administered subcutaneously (sub-Q) immediately before starting the operation. During surgery, sheep were surgically instrumented with vascular catheters in the common pulmonary artery via the right jugular vein, the abdominal aorta via the right femoral artery, and the left atrium via the left thoracotomy at the level of the fifth intercostal space to conduct hemodynamic monitoring. After the surgery, sheep were allowed to recover 5 to 7 days with free access to food and water. Buprenorphine was given when needed for postsurgical analgesia.

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Smoke inhalation and third-degree skin burn

After the surgical recovery, catheterized sheep were exposed to smoke inhalation with and without third-degree skin burn. Before the injury, baseline values of hemodynamics were obtained by connecting catheters to pressure transducers (model PX4X4, Baxter Edwards Critical Care Division, Irvine, Calif) in the healthy state. Prior to injury, sheep were randomly assigned to one of three groups: sham group (surgery, but no injury), smoke only (48 breaths of cooled cotton smoke), and combined smoke inhalation and third-degree skin burn (48 breaths of cooled cotton smoke and third-degree skin burn over 40% TBSA). Prior to inducing inhalation injury and third-degree skin burn, all sheep underwent tracheostomy (10 mm diameter, Shiley, Irvine, Calif), by published procedures (10). All injuries were performed under deep anesthesia (2–5% isoflurane) and analgesia (0.005–0.01 mg/kg of buprenorphine SR). The smoke used for injury was prepared by burning 40 g of cotton towels (9). Smoke inhalation was induced by insufflation of 48 breaths of cooled cotton smoke via a tracheostomy tube using a modified bee smoker. Arterial blood gas samples were collected and analyzed using a blood gas analyzer (RAPIDPoint 500 System, Siemens Healthcare Diagnostics, Tarrytown, NY) to determine carboxyhemoglobin (Co-Hb) levels, immediately after every four sets of 12 breaths of smoke insufflation. To ensure comparable injury between all injury groups, the temperature of the smoke was not allowed to exceed 40°C (9). The temperature of the smoke was measured by inserting a Swan-Ganz catheter (model 131F7, Edwards Critical Care Division, Irvine, Calif) into a modified endotracheal tube, connected to the modified bee smoker. Sheep in the smoke inhalation and combined injury groups received the same 48 breathes of cooled cotton smoke inhalation as described above, and were additionally subjected to third-degree skin burn over 40% of the TBSA via Bunsen burner (11).

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Postinjury care

Following injury, sheep were transferred to the TICU, and immediately placed on mechanical ventilation (Hamilton-G5, Hamilton Medical, Switzerland) support using APVcmv mode with positive end-expiratory pressure set at 5 cm H2O and monitored for 48 h in a conscious state. Tidal volume was maintained at 12 mL/kg and a respiratory rate of 20 breaths per minute. To accelerate dissociation of carbon monoxide from hemoglobin, sheep received 100% oxygen for the first 3 h following injury. Additionally, arterial oxygen tension was maintained above 95 mm Hg by adjusting the fraction of inspiratory oxygen. Hemodynamics measurements (mean arterial pressure (MAP); central venous pressure (CVP); mean pulmonary artery pressure (MPAP); and left atrium pressure (LAP)) were recorded on a monitor with graphics and digital displays (MP30, Philips, Andover, Mass). Pulmonary function, blood for the preparation of serum, and blood gas exchange were recorded every 6 h in the conscious state. All hemodynamics were recorded while sheep were calm and at a standing position. To ensure recording was taken at the same position every 6 h, hemodynamics were recorded after the transducer was placed at the olecranon joint on the frontal leg while the sheep were standing.

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Fluid resuscitation

To compensate for the severe fluid loss, fluid resuscitation using lactated Ringer's solution was calculated using the Parkland formula. To ensure accurate measurement of fluid intake during the 48-h monitoring postinjury, sheep were allowed free access to food but not water.

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Tissue collection

Forty-eight hours after smoke inhalation with and without third-degree skin burn, sheep were euthanized by IV administration of xylazine (0.2 kg/mg), ketamine (10–15 mg/kg), and buprenorphine (0.005–0.01 mg/kg). Sheep brains were collected and divided in half by a sagittal cut down the longitudinal fissure; one-half was fixed in 10% buffered formalin, and the other one-half was snap-frozen in liquid nitrogen and stored at −80°C. After gross neuropathological examinations, brain areas collected were the frontal cortex, basal ganglia, amygdala, hippocampus, thalamus, cerebellum, pons, and pituitary gland. Additionally, blood was collected for preparing serum for analysis every 6 h after induction of injury. One whole sheep brain per group was randomly selected for gross neuropathological representative examples. Briefly, whole brains were fixed for 3 weeks in 10% buffered formalin and then sliced in coronal sections (0.5 inches thick).

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Luxol Fast Blue (LFB) counterstained with hematoxylin and eosin (H&E)

To assess BBB dysfunction, any structural changes to blood vessel integrity, 4 to 5 μm sections were incubated in LFB/solvent blue 38 (Sigma Chemical, St. Louis, Mo), rinsed in 95% alcohol with agitation, and washed in running tap water for 5 min. Sections were differentiation by dipping in 0.05% Lithium carbonate (J.T. Baker Chemical Co, Phillipsburg, NJ) and 70% alcohol. The sections were counterstained with H&E (Varistain Gemini automatic stainer, Thermo-Fisher Scientific, Waltham, Mass).

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Periodic acidic Schiff (PAS)

The integrity of the basement membranes of blood vessels was evaluated by staining with Periodic acid–Schiff (PAS). The sections were incubated in 0.5% periodic acid (Fisher Scientific, Fair Lawn, NJ), treated with Schiff Reagent solution (Fischer Scientific, Fair Lawn, NJ), and counterstained with Mayer's hematoxylin (Polyscientific R&D Corp, Bay Shore, NY). Slides were rinsed clean with 0.15% acid alcohol (dH20, 100% alcohol, 6 mL HCl).

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Martius scarlet blue (MSB)

Sections were incubated in Weigert's Hematoxylin (Polyscientific R&D Corp, Bay Shore, NY) for 15 min and treated with 1% acid alcohol. The sections were rinsed in 95% alcohol with agitation and stained in Martius (Pfaltz & Bauer, Waterbury, Conn) for 2 min at room temperature. The sections were then stained in brilliant crystal 6R for and incubated with 1% phosphotungstic acid (Thermo-Fisher Scientific, Waltham, Mass) and rinsed in running tap water. This was followed by staining with 0.5% aniline blue (Thermo-Fisher Scientific, Waltham, Mass).

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Immunohistochemistry

To confirm neurovascular dysfunction of the BBB, 4–5 μm thick coronal sections were stained with mouse anti-albumin antibody (1:50; Abcam Cambridge, Mass). Slides underwent antigen retrieval by incubating in Tris/EDTA pH 9 (Sigma Chemical, St. Louis, Mo) before staining.

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Quantification

The total area of positive staining of the PAS and MSB sections were quantified using the Keyence BZ-X700 all-in-one fluorescence microscope (Itasca, Ill). The counts of microhemorrhaging, neutrophils, normal, dilated, and ruptured blood vessels were completed by using ImageJ software (NIH). All quantifications were performed on at least ten 10× magnification images taken at random throughout the field of view, by an investigator who was blinded to the treatments.

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Statistical analysis

GraphPad Prism 7 (La Jolla, Calif) was used to perform statistical analysis. Data are expressed as the mean ± standard error of the mean (SEM). Differences between groups were determined by one-way ANOVA and Tukey multiple comparison tests. Differences were considered significant for P < 0.05.

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RESULTS

Smoke inhalation with and without third-degree skin burn results in an increase of dilated and congested blood vessels throughout the brain

Gross anatomical analysis of brains from sheep subjected to smoke inhalation with- and without third-degree skin burn injury revealed numerous neuropathological changes, e.g., enlarged ventricles, bleeding in the lateral ventricles, rare cases, rupture of the lateral ventricles resulted in bleeding in the brain parenchyma, hemorrhagic infarcts, and macrohemorrhaging (Supplemental data, Figure 1, http://links.lww.com/SHK/A760).

Analysis of the partial pressure oxygen in the arterial blood (PaO2) during inhalation injury revealed no differences in hypoxia between the groups (Supplemental data, Figure 2, http://links.lww.com/SHK/A761). Thus suggesting that hypoxia is not the underlying cause of the gross CNS pathology observed following injury. Sheep were monitored during the first 48-h after injury in a conscious state in order to eliminate the possible effects of hemodynamic changes and hypoxia itself on the CNS pathology following injury. There were no significant changes in hemodynamics during the 48-h experimental period in cardiac output, heart rate, mean arterial pressure, and temperature (Supplemental data, Figure 2, http://links.lww.com/SHK/A761).

In order to characterize blood vessel dysfunction, 4–5 μm sheep brain sections of the frontal cortex, basal ganglia, amygdala, hippocampus, pons, cerebellum, and pituitary were stained with Luxol Fast Blue and Hematoxylin & Eosin (LFB/HE). Numerous dilated and congested blood vessels were visible in all areas examined (Fig. 1). Quantification analyses showed a significant increase in the number of dilated blood vessels following smoke inhalation with and without third-degree skin burn injury as compared with sham in all brain areas examined (sham vs. smoke inhalation injury: frontal cortex P < 0.01, basal ganglia P < 0.0001, amygdala P < 0.01, hippocampus P < 0.01, and cerebellum P < 0.001; sham vs. smoke inhalation with third-degree skin burn injury: frontal cortex P < 0.0001, basal ganglia P < 0.0001, amygdala P < 0.0001, hippocampus P < 0.001, and cerebellum P < 0.0001) (Fig. 1). There was also a significant increase in dilated vessels between the smoke inhalation group and the combined smoke inhalation with third-degree burn (frontal cortex P < 0.0001, basal ganglia P < 0.05, amygdala P < 0.05, and cerebellum P < 0.05) (Fig. 1).

Fig. 1

Fig. 1

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Smoke inhalation with and without third-degree skin burn results in an increase of the percent total dilated vessels versus total blood vessels throughout the brain

There was an increase in vessels that become dilated and congested following injury in the brain regions of interest (sham vs. smoke inhalation injury: basal ganglia P < 0.0001, amygdala P < 0.01, hippocampus P < 0.01, and cerebellum P < 0.0001; sham vs. smoke inhalation with third-degree skin burn injury: frontal cortex P < 0.0001, basal ganglia P < 0.0001, amygdala P < 0.0001, hippocampus P < 0.0001, and cerebellum P < 0.0001) (Fig. 2A). There was also a significant increase between the two injury groups (smoke inhalation injury vs. smoke inhalation with third-degree skin burn injury: frontal cortex P < 0.001, basal ganglia P < 0.0001, amygdala P < 0.001, hippocampus P < 0.05, and cerebellum P < 0.001) (Fig. 2A).

Fig. 2

Fig. 2

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Smoke inhalation with and without third-degree skin burn causes a significant decrease in the number of normal blood vessels

We also analyzed the percent normal blood vessels in each region (Fig. 2B). There was a significant decrease in the percent normal blood vessels following smoke inhalation injury and the combined smoke inhalation with third-degree skin burn as compared with sham (sham vs. smoke inhalation injury: frontal cortex P < 0.05, basal ganglia P < 0.0001, amygdala P < 0.001, hippocampus P < 0.001, cerebellum P < 0.01; sham vs. smoke inhalation with third-degree skin burn injury: frontal cortex P < 0.0001, basal ganglia P < 0.0001, amygdala P < 0.0001, hippocampus P < 0.0001, and cerebellum P < 0.0001). There was a significant decrease in the percent normal blood vessels between the smoke inhalation injury and the combined smoke inhalation with third-degree skin burn group (frontal cortex P < 0.0001, basal ganglia P < 0.0001, amygdala P < 0.001, hippocampus P < 0.05, and cerebellum P < 0.01) (Fig. 2B).

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The integrity of blood vessels basement membrane is lost following smoke inhalation with and without third-degree skin burn

The basement membranes of blood vessels are composed of several polysaccharides and glycoproteins such as perlecans, fibronectins, and dystroglycan. To determine whether the basement membranes of the dilated and congested blood vessels seen in Figure 1 were damaged following injury, we examined the integrity of the basement membrane using the PAS staining.

Damaged blood vessels were observed in all brain regions of interest (Fig. 3). A significant increase in damaged blood vessels following smoke inhalation injury and the combined smoke inhalation with third-degree skin burn was observed as compared with sham in the frontal cortex (P < 0.0001, smoke inhalation injury and P < 0.001, smoke inhalation with third-degree skin burn), basal ganglia (P < 0.0001, smoke inhalation injury and P < 0.001, smoke inhalation with third-degree skin burn), amygdala (P = 0.0862, smoke inhalation injury and P < 0.001, smoke inhalation with third-degree skin burn), hippocampus (P < 0.001, smoke inhalation injury and P < 0.01, smoke inhalation with third-degree skin burn), pons (P < 0.0001, smoke inhalation injury and P < 0.001, smoke with third-degree skin burn), cerebellum (P < 0.0001 smoke inhalation injury and P < 0.001, smoke with third-degree skin burn), and pituitary gland (P < 0.05, smoke inhalation with third-degree skin burn), (Fig. 3). The combined smoke inhalation with third-degree skin burn group had a statistically significant increase in damaged blood vessels in the amygdala (P < 0.05, sham vs. smoke with third-degree skin burn injury) (Fig. 3).

Fig. 3

Fig. 3

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Smoke inhalation with and without third-degree skin burn results in dilated blood vessels that rupture in the brain

The LFB/H&E revealed numerous congested and dilated vessels (Fig. 1) that rupture. An increase in ruptured vessels following the smoke inhalation injury and the combined smoke inhalation with third-degree skin burn injury as compared with sham was observed in the frontal cortex (P = 0.0715, smoke inhalation injury and P < 0.01, smoke inhalation with third-degree skin burn injury) basal ganglia (P < 0.0001, smoke inhalation injury and P < 0.001, smoke inhalation with third-degree skin burn injury) amygdala (P < 0.001, both injury groups), hippocampus (P < 0.05, smoke inhalation injury), and the cerebellum (P < 0.01, smoke inhalation with third-degree skin burn injury) (Fig. 4).

Fig. 4

Fig. 4

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Smoke inhalation with and without third-degree skin burn results in an increase in the percent of ruptured blood vessels in the brain

We determined that there was an increase in total vessels that rupture following injury in brain regions of interest. Quantification analysis showed an increase in the percent total ruptured vessels compared to sham following smoke inhalation injury and the combined smoke inhalation with third-degree skin burn injury groups (sham vs. smoke inhalation injury: basal ganglia P < 0.001, amygdala P < 0.01, hippocampus P = 0.0973, and cerebellum P = 0.2290; sham vs. smoke inhalation with third-degree skin burn: frontal cortex P < 0.01, basal ganglia P < 0.0001, amygdala P < 0.001, hippocampus P = 0.1349, and cerebellum P < 0.001 (Fig. 5A).

Fig. 5

Fig. 5

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Smoke inhalation causes an increase in dilated vessels that rupture in the brain

We measured how many of the dilated vessels ruptured following smoke inhalation injury and the combined smoke inhalation with third-degree skin burn. The percent of total ruptured blood vessels that were dilated increased following smoke inhalation injury and with the combined smoke inhalation with third-degree skin burn as compared with sham (sham vs. smoke inhalation injury: frontal cortex P < 0.01, basal ganglia P < 0.0001, amygdala P < 0.0001, hippocampus P < 0.05, and cerebellum P < 0.05; sham vs. smoke inhalation with third-degree skin burn: frontal cortex P < 0.05, basal ganglia P < 0.001, amygdala P < 0.01, and cerebellum P < 0.01 (Fig. 5B). There was a significant increase in the percent total ruptured vessels between the injury groups in the basal ganglia, P < 0.05 and amygdala, P < 0.01 (Fig. 5B).

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Smoke inhalation with and without third-degree skin burn results in microhemorrhaging in the brain

LFB/H&E staining showed severe microhemorrhaging following smoke inhalation with and without third-degree skin burn (Fig. 6). A significant increase in microhemorrhaging following smoke inhalation injury and the combined smoke inhalation with third-degree as compared with sham was observed in the frontal cortex (P < 0.01, smoke inhalation injury and P < 0.01, smoke inhalation with third-degree skin burn injury), basal ganglia (P < 0.01, smoke inhalation injury and P < 0.01, smoke inhalation with third-degree skin burn injury), amygdala (P = 0.7950, smoke inhalation injury and P = 0.1486, smoke inhalation with third-degree skin burn injury), hippocampus (P < 0.05, smoke inhalation injury), and cerebellum (P = 0.3185, smoke inhalation injury and P = 0.0550, sham vs. smoke inhalation with third-degree skin burn) (Fig. 6). The pituitary is a glandular tissue. Therefore, it was difficult to distinguish the area of individual microbleeds. Consequently, we measured the total RBCs in the tissue (Fig. 6). There was a significant increase in total RBCs in the pituitary following smoke inhalation injury and the combined smoke inhalation with third-degree skin burn (P < 0.05, sham vs. smoke inhalation injury and P < 0.05, sham vs. smoke inhalation with third-degree skin burn injury).

Fig. 6

Fig. 6

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Confirmation of microhemorrhaging following smoke inhalation with and without third-degree skin burn by positive MSB staining

To further characterize the microhemorrhaging observed in the LFB/H&E staining, we analyzed fibrin(ogen) deposition into the brain parenchyma using MSB staining (Fig. 7). There was an increase in fibrin(ogen) deposition in the frontal cortex (P = 0.2461, smoke inhalation injury and P < 0.001, smoke inhalation with third-degree skin burn injury), basal ganglia (P < 0.01, smoke with third-degree skin burn injury), amygdala (P < 0.01, smoke inhalation injury and P < 0.001, smoke inhalation with third-degree skin burn injury), hippocampus (P = 0.0655, smoke inhalation injury and P = 0.1010, smoke inhalation with third-degree skin burn injury), pons (P = 0.05, smoke inhalation injury and P < 0.01, smoke inhalation with third-degree skin burn injury), cerebellum (P < 0.01, smoke with third-degree skin burn injury), and pituitary gland (P < 0.0001, smoke inhalation with third-degree skin burn injury) (Fig. 7). There was a significant increase in fibrin(ogen) deposition between the injury groups in the frontal cortex (P < 0.05, smoke inhalation injury vs. smoke inhalation with third-degree skin burn), basal ganglia (P < 0.05, smoke inhalation injury vs. smoke inhalation with third-degree skin burn), and pituitary gland (P < 0.01, smoke inhalation injury vs. smoke inhalation with third-degree skin burn) (Fig. 7).

Fig. 7

Fig. 7

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Smoke inhalation with and without third-degree skin burn results in microhemorrhaging in the brain confirmed by positive albumin staining

Immunohistochemistry was used to assess BBB dysfunction by examining albumin extravasation in the brain parenchyma (Fig. 8). In all brain regions examined, there was an increase in albumin staining in the smoke inhalation injury and the combined smoke inhalation with third-degree skin burn injury groups as compared with sham (sham vs. smoke inhalation with third-degree skin burn: frontal cortex P < 0.05 and basal ganglia P < 0.01 (Fig. 8). In the basal ganglia, there was a significant increase in albumin deposition in the smoke inhalation with third-degree skin burn group as compared with smoke inhalation injury (P < 0.05, smoke inhalation injury vs. smoke inhalation with third-degree skin burn injury) (Fig. 8). There was an increase in albumin staining in the other regions of interest. However, due to high variability, the increase was not statistically significant.

Fig. 8

Fig. 8

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Smoke inhalation with and without third-degree skin burn results in neutrophil infiltration in the brain

Neutrophil infiltration was observed in all regions of interest in the LFB/H&E staining (Fig. 9). There was a significant increase in neutrophils following smoke inhalation injury and the combine smoke inhalation with third-degree skin burn in the frontal cortex (P < 0.01, sham vs. smoke inhalation injury and sham vs. P = 0.1255 smoke with third-degree skin burn), basal ganglia (P = 0.1669, sham vs. smoke inhalation injury and sham vs. P = 0.1749 smoke inhalation with third-degree skin burn), amygdala (P = 0.1661, sham vs. smoke inhalation injury and P < 0.05, sham vs. smoke inhalation with third-degree skin burn), hippocampus (P = 0.1854, sham vs. smoke inhalation injury), cerebellum (P = 0.1328, sham vs. smoke inhalation injury), and pituitary gland (P = 0.1356, sham vs. smoke inhalation injury) (Fig. 9).

Fig. 9

Fig. 9

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DISCUSSION

Although the pathophysiology of smoke inhalation injury has been extensively studied, little is known about the acute and long-term effects of smoke inhalation on the CNS. As of today, CNS dysfunctions following smoke inhalation injury have been studied by only two published clinical case reports. These studies, although limited to only a few patients, clearly indicate that neurological complications such as a persistent headache, memory loss, and paresthesia occur following smoke inhalation injury alone (3). Additionally, one case report concluded that acute smoke inhalation injury results in a long-term global decrease in metabolic activity and degeneration of several areas of the brain (8). Clearly, CNS dysfunction following smoke inhalation with and without third-degree skin burn is a neglected field of investigation, and a better understanding of these pathological changes will allow us to effectively treat patients who have suffered smoke inhalation with and without third-degree skin burn. This is particularly important because recent advances in clinical care and the development of novel treatments have greatly increased the survival of burn injury and smoke inhalation injury patients, thus warranting the need for follow-up studies aimed at determining possible pathological alterations of the CNS and associated cognitive dysfunctions.

In this report we used a well-established ovine model that reproduces injuries suffered by victims of fire such as firefighters, factory, and construction workers (2), and showed, for the first time, that smoke inhalation, regardless of the presence of third-degree skin burn, leads to diffuse loss of blood-brain barrier integrity resulting in massive bleeding throughout the brain without evidence of changes in hypoxia or hemodynamics. Although the model we used does not allow studying specific cognitive deficits due to lack of established learning and behavioral memory tests in sheep, the literature clearly demonstrates the existence of a strong connection between the diffuse loss of blood-brain barrier integrity and clinical manifestations of neurological dysfunction. A detailed discussion of our findings and their clinical relevance is discussed below.

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Smoke inhalation with and without third-degree skin burn caused bleeding in the lateral ventricles and macrohemorrhaging throughout the brain

Our data show that acute smoke inhalation with and without third-degree skin burn produces intraventricular hemorrhaging resulting in enlarged ventricles that, in some cases, ruptured causing bleeding in the brain parenchyma (Supplemental data, Figure 1, http://links.lww.com/SHK/A760).

Several studies demonstrate that intraventricular hemorrhaging can lead to several life-threatening complications such as decreased level in consciousness, particularly with hemorrhaging in the reticular activating system, that control sleep and arousal states (waking, asleep, and asleep with dreaming) and in the thalamus (12, 13). Several randomized controlled stroke studies showed that bleeding in the lateral ventricles increases mortality by 50 to 80%, decreases the level of consciousness, results in a poorer functional outcome, and increases morbidity (12, 14, 15). In particular, intraventricular hemorrhaging results in blood clots that can block cerebrospinal fluid (CSF) limiting cerebral perfusion, resulting in obstructive (communicating) hydrocephalus and contributing to the mass effect and cerebral edema in these patients (12). Moreover, the persistence of blood clots and blood breakdown products in the lateral ventricles cause decreased blood clot resolution due to the presence of coagulation and fibrinolytic pathways leading to accumulation of blood breakdown products, and consequently to an inflammatory reaction (12). This is relevant because previous studies using animal models of intraventricular hemorrhage demonstrated that such inflammatory reaction could affect long-term cognitive function (12, 14). These findings were also supported by Hutter et al. who demonstrated significant deficits in neuropsychological testing in stroke patients with subarachnoid and intraventricular hemorrhaging as compared to patients without intraventricular hemorrhage (12, 16). Together with these findings, our data provide strong support to the notion that smoke inhalation with and without third-degree skin burn can lead to cognitive deficits due to bleeding in the lateral ventricles.

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Smoke inhalation with and without third-degree skin burn caused BBB dysfunction characterized by congested and dilated blood vessels and blood vessels that rupture throughout the brain

Our data show that smoke inhalation with and without third-degree skin burn leads to congestion of red blood cells (RBCs) in the lumen resulting in dilation of arterial blood vessels in the frontal cortex, basal ganglia, amygdala, hippocampus, pons, cerebellum, and pituitary gland (Fig. 1). Further, using PAS staining we demonstrated that damage resulting in weakening of the blood vessels basal membrane is likely the underlying cause of BBB dysfunction following smoke inhalation alone or in combination with third-degree skin burn (Fig. 3).

We further demonstrated that following smoke inhalation, alone or in combination with third-degree skin burn, microvascular dysfunction caused by the obstruction of the blood vessels leads to a significant increase in ruptures suggesting that toxic chemical(s) in the smoke could damage and weaken the blood vessels wall (Fig. 4). Specifically, we found that the percentage of dilated vessels that ruptured was higher after smoke inhalation alone in the frontal cortex, basal ganglia, amygdala, and hippocampus as compared to the combined smoke inhalation with third-degree skin burn. The cerebellum was the only region where the combined smoke inhalation with third-degree skin burn resulted in higher percentage of rupturing dilated vessels.

Bleeding in the brain causes a series of deleterious events leading to secondary brain injuries such as inflammation, oxidative stress, hypermetabolism, excitotoxicity, and hematoma cytotoxicity (17). Specifically, and of relevance to our study, several clinical studies demonstrated that bleeding in the basal ganglia is associated with motor weakness, attention and language deficits, significant impairments in, learning, executive and visual and spatial functions depending on the location of the ruptured blood vessels (18, 19). In fact, due to the high level of connectivity to different brain regions, the basal ganglia control motor functions, limbic functions (emotions), and executive functions (attention, memory, implicit learning, and habit formation) (20).

In the hippocampus, the brain region that plays a major role in consolidating short-term memories into long-term memories, bleeding has been associated with memory impairments, difficulty in word finding, hemiparesis, and attention deficits (21).

The pons, a component of the brain stem, plays a role in controlling respiratory function, sleep-wake cycle, and consciousness (22). Strokes in the pons are characterized by neurological deficits, respiratory failure, headache, vertigo, and disturbances of consciousness (23). Additionally, a clinical review of 78 patients with brainstem strokes by D’aes and Mariën reported patients suffered from impairment of limbic or cortical areas presenting as cognitive symptoms such as deficits in attention, executive function, intellectual capacity, memory, language, visuo-spatial skills (24).

In the cerebellum, the brain region that coordinates voluntary movements, acute infarctions due to rupturing of blood vessels are associated with several neurological complications such as declining level of consciousness, coma, ataxia, headache, dysarthria, vertigo, poor balance, difficulty walking, obstructive hydrocephalus, and brain stem compression due to hemorrhagic mass effect (25).

Following large burns, patients suffer from a hypermetabolic response associated with a severely altered hormone profile due to hypothalamic-pituitary-organ axis dysfunction (26). During the acute phase of hypothalamic-pituitary-organ axis dysfunction, the pituitary gland is actively secreting hormones; however, due to the development of target-organ resistance, peripheral hormone levels are low. The endocrine axis is suppressed by the hypothalamus during the long-term phase resulting in low peripheral hormone levels. Several clinical trials aimed to correct hormonal dysbalances following critical injuries were unsuccessful (26). Vanhorebeek et al., 2006 believed this failure was due to the lack of knowledge of the pathophysiological mechanism (27). Our data suggest that changes in the hypothalamic-pituitary-organ axis dysfunction could be linked to microhemorrhaging in the pituitary gland.

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Smoke inhalation with and without third-degree skin burn results in microhemorrhaging confirmed by positive fibrin(ogen) staining in the brain parenchyma

The extravasation of RBCs and blood proteins into brain parenchyma was further exacerbated by microhemorrhaging following smoke inhalation with and without third-degree skin burn. There was a significant increase in microhemorrhaging in the frontal cortex, basal ganglia, and the pituitary gland following smoke inhalation with and without third-degree skin burn as compared to sham (Fig. 6). Following smoke inhalation injury, there was a significant increase in microhemorrhaging in the hippocampus. In the amygdala and cerebellum, there was an increase in microhemorrhaging, with the combined smoke inhalation injury with third-degree skin burn having the highest increase (Fig. 6).

We used MSB staining (Fig. 7) to confirm microhemorrhaging throughout the brain following injury. Fibrin(ogen) is a 340-kD glycoprotein normally present in the blood plasma and absent in the brain parenchyma. Using MSB staining, in this report, we found a significant increase in fibrin(ogen) deposition following smoke inhalation alone in the amygdala and pons (Fig. 7). In the frontal cortex, basal ganglia, amygdala, pons, cerebellum, and pituitary gland, there was a significant increase in fibrin(ogen) deposition following the combined smoke inhalation with third-degree skin burn injury as compared to sham controls (Fig. 7). We also found significantly higher fibrin(ogen) deposition in the combined smoke inhalation with third-degree skin burn injury when compared with smoke inhalation injury in the frontal cortex, basal ganglia and pituitary gland (Fig. 7).

Extravasation of fibrin(ogen) from the blood circulation into the brain parenchyma results in increased neurovascular damage, neuroinflammation, neuronal degeneration, and cognitive decline (28, 29). Several studies have demonstrated microhemorrhaging is associated with motor disturbances and cognitive impairment in information processing, executive functions, and slower motor speeds (30, 31). Additionally, fibrin(ogen) deposition into the parenchyma has been shown to activate CNS innate and adaptive immune responses by interacting with the CD11b/CD18 integrin receptor on microglia, macrophages, and neutrophils (28, 32).

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Smoke inhalation with and without third-degree skin burn results in neutrophil infiltration into the brain

Circulating neutrophils are the first to migrate to areas of infection or invading microbes (33). We found that following smoke inhalation, alone and in combination with third-degree skin burn, there was an increase in granulated polymorphonuclear cells (PMNs), such as neutrophils, throughout the brain. Notably, we found a higher number of infiltrating neutrophils in the frontal cortex, basal ganglia, amygdala, hippocampus, cerebellum, and the pituitary gland following smoke inhalation injury alone (Fig. 9). We believe neutrophil infiltration is highest in this group because it is not being sequestered to the periphery due to third-degree skin burn. On the other hand, reduced neutrophil infiltration following smoke inhalation with third-degree skin burn could be due to impairment of the immune system. In fact, Cheng et al. (34) reported that impaired immune function occurs following a 10% decrease in body mass, such as the one known to occur after large skin burns (30–40% TBSA) as a result of profound hypermetabolic and inflammatory response (26).

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CONCLUSION

In this report we demonstrate, for the first time that smoke inhalation, alone or in combination with third-degree skin burn injury, produces massive bleeding and profound pathological changes in the brain that are not secondary to changes in hypoxia or hemodynamics. Moreover, our results indicate that a well-characterized ovine model of smoke inhalation with and without third-degree skin burn is not only a good model to investigate pulmonary pathophysiology, but also a useful model to elucidate acute damages to the CNS following smoke and burn injury.

In conclusion, our data demonstrated important pathological alterations in the CNS after smoke injury and lay the foundation for future studies aimed at further characterizing the contributing factors leading to progressive neurological deficits observed after acute exposure to smoke inhalation. These findings have a high impact on the field because, by identifying smoke inhalation with and without third-degree skin burn as an underlying cause for CNS dysfunction, strongly points to the need to include neurological examinations in the standard operating protocol for these patients.

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Acknowledgments

The authors thank the members of Dr. Micci's laboratory: Elizabeth Bishop, Austin Cody Grant, Jutatip Guptarak, and Emanuele Mocciaro for providing training on the Keyence microscope and assisting with the data analysis. The authors thank all the members (Ayotola Akinola, Randi Bolding, Haley Flowers, Kang Ko, Christina Nelson, Cynthia Moncebiaz, Randy Salsbury, Ryan Scott, and Ashley Smith) of the Translational Intensive Care Unit for technical assistance with the sheep surgeries, post-surgery monitoring and collection of hemodynamic data, the Research Histopathology Core (Kenneth Escobar) for histopathology training, Dr. Owen Hamill for assisting with the experimental design, and Dr. Gerald Campbell for neuropathology training.

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Keywords:

Blood vessel dilation; blood vessels; blood–brain barrier dysfunction; brain pathology; microhemorrhage; neurological function; skin burn injury; smoke inhalation

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