The present study was approved by the Massachusetts General Hospital Subcommittee on Research Animal Care. Sprague–Dawley viral-free rats, all in the growing phase, weighing between 185 and 225 g, were obtained from Charles River Laboratories (Wilmington, MA, USA).

The animals were anesthetized by intraperitoneal ketamine (90 mg/kg) (Abbott Laboratories, Chicago, IL, USA) and xylazine (10 mg/kg; Burns Veterinary Supply Inc., Rockville Centre, NY, USA) while breathing room air. Throughout the experiment, the animals were placed in a supine position on a heating blanket and the body temperature was monitored with a rectal probe. PE 240 tubing (outer diameter, 2.42 mm; internal diameter, 1.67 mm; Becton Dickson Infusion Therapy System Inc., Sandy, UT, USA) was inserted into the trachea and connected to a Harvard apparatus ventilator (model 55-7058; Harvard Apparatus, Holliston, MA, USA). The rats were then ventilated with a tidal volume of 7 ml/kg with a rate of 85 to 100 breaths per minute.

The end-tidal carbon dioxide pressure was monitored intermittently by a microcapnograph (Columbus Instruments, Columbus, OH, USA), and was maintained between 35 and 45 mmHg by adjusting the ventilator respiratory rate. The volume was increased by 5 ml/min to correct the air loss from the sample flow adaptor during monitoring of the end-tidal carbon dioxide. The peak inspiratory airway pressure was measured every 30 minutes with a pressure transducer amplifier (Gould Instrument System, Valley View, OH, USA) connected to the tubing at the proximal end of the tracheostomy.

The mean arterial pressure was measured every 30 minutes during mechanical ventilation using the same pressure transducer amplifier connected to PE 10 tubing (outer diameter, 0.61 mm; inner diameter, 0.28 mm; Becton Dickson Infusion Therapy System Inc., Sandy, Utah, USA) ending in the common carotid artery. During the period of ventilator use, intraperitoneal ketamine 0.05 mg/g and xylazine 0.005 mg/g were administered every 30 minutes, and 0.9% NaCl was infused, as needed, to maintain systolic blood pressure >90 mmHg. Harvesting of lung and bronchoalveolar lavage (BAL) was performed after 4 hours of mechanical ventilation.

Sprague–Dawley rats were randomly divided into four groups: nonventilated control rats; mechanically ventilated rats with lipopolysaccharide (LPS) infusion as a model of sepsis; mechanical ventilation plus LPS infusion with HMW HA (1,600 kDa) pretreatment; and mechanical ventilation plus LPS infusion with LMW HA (35 kDa) pretreatment. Rats were mechanically ventilated with a low tidal volume (7 ml/kg) (n = 5 or 6 rats/group) for 4 hours. Rats received 3 ml of 0.35% of 1,600 kDa or 35 kDa (Genzyme Corp., Cambridge, MA, USA) pretreatment via the intraperitoneal route 18 hours prior to the beginning of study.

All HA preparations were sterile and were protein free and LPS free, to avoid known confounding effects of HA 10. The size and amount of HA was chosen based on previous work that found HMW HA (>780 kDa) given intraperitoneally 18 hours before injection of concavalin protected against concavalin-induced liver toxicity. A dose response was found in this model, and 0.35% HMW HA was most effective 11. We have shown that 1,600 kDa HA is the predominant size in normal rat lung 7. Based on these findings, we used 0.35% of 1,600 kDa given intraperitoneally 18 hours prior to LPS infusion.

Rats received either 1 mg/kg Salmonella typhosa LPS (Lot 81H4018; Sigma Chemical Co., St Louis, MO, USA) or an equivalent volume of normal saline as control via the carotid artery. We have previously found arterial injection of LPS with mechanical ventilation to cause acute lung injury within 4 hours of injection. After 1 hour of spontaneous respiration to allow for development of a septic response, ventilation was begun. We used an established rodent model of mechanical ventilation as previously described 121314. Rats were sacrificed with an overdose of pentobarbital after 4 hours of ventilation. The left lung was lavaged with normal saline for measurement of cell counts and cytokines, macrophage inflammatory protein-2 (MIP-2) and TNFα. The right lung was flash frozen for the myeloperoxidase assay, for extraction of RNA for the measurement of HA synthase, and for determining the gene expression of cytokines, MIP-2 and TNFα. Separate groups of animals were used for determination of lung pathology.

Rats were ventilated in the same manner as described for the pretreatment with HA model. The 0.35% concentration of 1,600 kDa HA was too viscous for intravenous injection. For the studies post acute lung injury treatment, we performed dose–response studies with 0.025%, 0.05% and 0.1% of 1,600 kDa HA starting at the time of initiation of ventilation. Intravenous infusion of 0.1% sodium carboxymethyl cellulose (CMC) – a positive charged carbohydrate prepared from carboxymethylation of cellulose with a molecular weight of 1 × 10⁵ to 7.5 × 10⁵ – was used as a control condition for charge at 500 μl/hour. The infusion was continued throughout the experiment.

The lungs were removed en bloc and tubing was inserted into the trachea and secured. The right lung was clamped at the bronchus to prevent the lavage fluid from entering the right lung. The left lung was lavaged with 2 ml of 0.9% normal saline three times. One millilitre of the pooled effluents was used for cytospin and subsequent cell differentials, and 100 μl was used for the total cell counts. The remaining effluents were centrifuged at 3,000 rpm for 10 minutes, after which the supernatants were frozen at -80°C for further measurement of cytokines.

Neutrophil counts in BAL fluid were used to measure migration of neutrophils into alveoli and airways 15. Total cell counts in BAL were performed using a hemocytometer. To measure cell differentials, the cells in the lavage fluid were fixed on glass slides with cytospin and were then stained with a hematologic stain kit (Fisher Diagnostics, Middletown, VA, USA).

Myeloperoxidase activity in lung parenchyma was used as a marker of total lung neutrophil sequestration, including neutrophils marginalized in the vasculature, and in the interstitium and alveoli 131516. Samples of the right lower lobe were obtained within a few minutes of death and were stored at -80°C. The right lower lobe was thawed on ice, weighed, and homogenized in 5 ml phosphate buffer (20 mM, pH 7.4). One milliliter of the homogenate was centrifuged at 10,000 × g for 10 minutes at 4°C. The resulting pellet was resuspended in 1.0 ml phosphate buffer (50 mM, pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (Sigma Chemical Co.). The suspension was subjected to three cycles of freezing (on dry ice) and thawing (at room temperature), after which it was sonicated for 40 seconds and centrifuged again at 10,000 × g for 5 minutes at 4°C.

The supernatant was assayed for myeloperoxidase activity by measurement of hydrogen peroxide-dependent oxidation of 3,3',5,5'-tetramethylbenzidine (Sigma Chemical Co.). In its oxidized form, 3,3',5,5'-tetramethylbenzidine was measured by spectrophotometer at 650 nm. The reaction mixture for analysis consisted of 25 μl tissue samples, 25 μl 3,3',5,5'-tetramethylbenzidine (final concentration, 0.16 mM) dissolved in dimethylsulfoxide, and 200 μl hydrogen peroxide (final concentration, 0.30 mM) dissolved in phosphate buffer (0.08 M, pH 5.4) prior to adding to the mixture. The reaction mixture was incubated for 3 minutes at 37°C and the reaction stopped by adding 1 ml sodium acetate (0.2 M, pH 3.0), after which absorbance at 650 nm was measured. The absorbance followed a linear relationship with the myeloperoxidase concentration, which in turn is an enzyme marker for leukosequestration. The absorbance (A₆₅₀) was reported as units (optical density) per gram of wet lung weight.

Rat MIP-2 and TNFα were measured in BAL fluid using a commercially available ELISA kit containing antibodies that were cross-reactive with rats and mouse MIP-2 (BioSource International, Inc., Camarillo, CA, USA). Each sample was run in duplicate according to the protocol provided by the manufacturer.

For isolation of total RNA, the lungs were homogenized in 1.5 ml Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and were isolated according to the manufacturer's protocol. Total RNA (1 μg) was reversely transcribed into cDNA using a Gene Amp PCR system 9600 (PerkinElmer Life Sciences, Boston, MA, USA), as previously described 17.

The following primers of MIP-2 were used: PCR forward primer, 5'-TCC TCA ATG CTG TAC TGG TCC-3' and reverse primer, 5'-ATG TTC TTC CTT TCC AGG TC-3'; TNFα forward primer, 5'-CAT GAT CCG AGA TGT GGA ACT-3' and reverse primer, 5'-TCA CAG AGC AAT GAC TCC AAA G-3'; and GAPDH (internal control) forward primer, 5'-AAT GCA TCC TGC ACC ACC AA-3' and reverse primer, 5'-GTA GCC ATA TTC ATT GTC ATA-3' (Sigma Chemical Co.).

The following cycling parameters were used: MIP-2, denaturation at 94°C for 5 minutes followed by 35 cycles of 94°C for 30 seconds, annealing at 50°C for 45 seconds, and extension at 72°C for 30 seconds, with a terminal extension at 72°C for 7 minutes; and for TNFα, denaturation at 94°C for 5 minutes followed by 40 cycles of 94°C for 30 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 1 minute, with a terminal extension at 72°C for 7 minutes.

Results were quantified using densitometry. The GAPDH and cytokine signal densitometry was measured for each group. The cytokine signal was normalized to GAPDH expression and expressed as a ratio to control. A minimum of three mRNA samples were analyzed for each group.

After 4 hours of mechanical ventilation, the rats were sacrificed and the lung and trachea were removed. The left lung was infused at a pressure of 30 cmH₂O with 10% buffered formalin, embedded in paraffin, sectioned at 4 μm thickness, and stained with hematoxylin and eosin. Ten randomly chosen fields in the parenchyma (without large airways) from the individual three lungs from each group were examined. Each of the pathological changes was scored on a scale of 0 to 3: 0 = alveolar filling, collapse or atelectasis (10×); 1 = inflammatory cell infiltration in the air space or vessel wall (20×); 2 = perivascular clubbing or swelling (10×); and 3 = alveolar hemorrhage or congestion (10×). The 10 randomly chosen fields at low power (10×) covered over 80% of the left lung. Because the injury was patchy, this technique gave an overview of the whole left lung. A higher power (20×) was needed to accurately identify inflammatory cell infiltration. The overall score was the sum of the average score for each category. Two subspecialists who were blinded to the treatment groups reviewed the degree of injury of each slides.

Analysis was performed using Statview 4.5 (SAS Institute Inc., Cary, NC, USA). All data are expressed as the mean ± standard error of mean. Analysis of variance for comparison of the different groups was used with significance set at P < 0.05. A significant analysis of variance was followed by a Fisher test for multiple comparisons between groups, significance set at P < 0.05.

For animals in the experiments involving HA pretreatment, the systolic pressure was maintained with infusion of saline as needed to maintain systolic blood pressure >70 mmHg to minimize the confounding effects of hypotension. The amount of saline infused did not differ among groups. The peak airway pressures for all animals were between 8 and 12 mmHg. The systolic blood pressure did not differ among groups. The heart rate was increased in animals exposed to LPS but was not significantly different between treatment groups (Table 1).

Rats receiving LPS had increased BAL neutrophils as compared with rats without LPS treatment. Pretreatment with HMW HA (1,600 kDa) decreased BAL neutrophils and the total lung neutrophil infiltrate with LPS. The results were similar with the use of 35 kDa HA (Figure 1). Lung neutrophil infiltration was confirmed with the myeloperoxidase assay (Figure 1). Only 1,600 kDa HA, and not 35 kDa HAcompletely blocked the increase in BAL monocytes. With LPS, 35 kDa HA only partially blocked BAL monocyte infiltration (Figure 1).

Rats with LPS had poor alveolar distention and collapse, had intense inflammatory cell infiltration in the interstitium, had thickened perivascular clubbing and had alveolar hemorrhage on pathology slides (Figure 2). The lung injury score of the rats receiving LPS was significantly greater compared with rats without LPS (Figure 3). We found that rats with LPS receiving 1,600 kDa HA pretreatment, but not 35 kDa HA pretreatment, had less evidence of lung injury on pathology and had significantly decreased lung injury scores (Figures 2 and 3). The inhibition of lung injury by 1,600 kDa HA and not 35 kDa HA correlated with 1,600 kDa HA inhibition of IL-1β.

In a comparisonn between rats with and without LPS at the same tidal volume, rats with LPS showed increased MIP-2 and TNFα mRNA and protein levels compared with rats without LPS. Rats receiving either 1,600 kDa HA or 35 kDa HA pretreatment showed less MIP-2 and TNFα production at the mRNA and protein level compared with rats with sepsis (Figures 4 and 5).

To further investigate the use of HMW HA infusion as a treatment for sepsis-induced lung injury, HMW HA (0.025%, 0.5% and 0.1%) was infused at 0.5 ml/hour starting at the time of ventilation. For these experiments, the maximum dose of LPS (3 mg/kg) that allowed survival of the animals was used. Saline was infused as needed to maintain a systolic pressure of about 70 mmHg to eliminate the confounding effects of hypotension. The systolic blood pressure was not significantly different between groups.

The heart rate was significantly higher in animals exposed to LPS, there were no significant differences between HMW HA-treated groups, and animals treated with CMC had a higher heart rate than those exposed to 0.1% HMW HA and than control animals (Table 2). All doses of HA and CMC increased the arterial oxygen pressure levels significantly (P < 0.05) above LPS-exposed animals (LPS, 51 ± 10 mmHg; LPS + 0.025% HA, 84 ± 7 mmHg; LPS + 0.05% HA, 67 ± 3 mmHg; LPS + 0.1% HA and LPS + 0.1% CMC, 75 ± 7 mmHg).

HMW HA inhibited neutrophil infiltration (Figure 6), MIP-2 mRNA expression (data not shown), and acute lung injury scores (Figure 7) in a dose-dependent manner. To evaluate whether this effect was secondary to the positive charge of HA or to the anti-inflammatory properties of HA, 0.1% CMC was infused at 0.5 ml/hour. CMC at an equal concentration to HMW HA (0.1%) partially blocked lung neutrophil infiltration (Figure 6) and lung injury (Figure 7), but not to the same extent as HMW HA – suggesting that the positive charge of HMW HA was at least partially responsible for the therapeutic effects.

Post-treatment with intraperitoneal HMW HA failed to protect against inhalation acute lung injury, and therefore was not used in the present study (data not shown)