Protective effects of hemoglobin vesicles on intestinal ischemia-reperfusion injury in a rat model of thoracic aortic occlusion

Abstract

Intestinal ischemia-reperfusion (I/R) injury is a major complication of aortic occlusion during resuscitative thoracotomy or resuscitative endovascular balloon occlusion of the aorta. We investigated whether targeted intra-arterial delivery of hemoglobin vesicles (HbVs) attenuates intestinal I/R injury in a rat thoracic aortic cross-clamping model. Male Wistar rats underwent 60 minutes of thoracic aortic occlusion followed by 60 minutes of reperfusion. Animals received intra-arterial HbV (1.25 mL/kg every 10 minutes; n = 5) or saline (n = 5) via the femoral artery; sham animals (n = 3) underwent instrumentation only. Compared with saline controls, HbV-treated rats demonstrated higher postreperfusion systolic blood pressure (56 ± 4 mm Hg vs 34 ± 8 mm Hg; P = .002) and lower plasma lactate levels (2.7 ± 0.4 mmol/L vs 7.5 ± 0.4 mmol/L; P < .001). Intestinal injury was significantly attenuated in the HbV group, with lower Chiu scores (median 2.0 vs 4.0; P = .018), reduced terminal deoxynucleotidyl transferase dUTP nick end labeling-positive area fraction (P = .003), and decreased inducible nitric oxide synthase-positive area fraction (P = .006). Targeted intra-arterial HbV administration significantly reduced early intestinal I/R injury in this exploratory proof-of-concept model and may represent a promising adjunctive oxygen-carrying strategy to protect distal organs during prolonged aortic occlusion.

Aortic occlusion during resuscitative thoracotomy or resuscitative endovascular balloon occlusion of the aorta is a lifesaving intervention for noncompressible torso hemorrhage.1, 2, 3 However, complete aortic cross-clamping inevitably produces severe distal ischemia and subsequent ischemia-reperfusion (I/R) injury, and the intestines are particularly vulnerable.4, 5, 6 Clinically, resuscitative endovascular balloon occlusion of the aorta is also associated with significant ischemic complications.⁷ The risk of distal organ injury increases with prolonged occlusion; therefore, experimental and clinical evidence emphasizes minimizing complete zone I occlusion time.8, 9, 10

Importantly, most current mitigation strategies seek to reduce the ischemic burden by modulating the duration or completeness of occlusion (eg, intermittent/partial approaches).1, 2, 3^,8, 9, 10 In contrast, the present approach is conceptually distinct: it aims to augment distal oxygen-carrying capacity during complete cross-clamping via targeted intra-arterial delivery of an oxygen carrier to vascular beds downstream of the clamp, rather than relying on systemic circulation or collateral flow.

Intestinal I/R injury triggers oxidative stress, epithelial apoptosis, barrier dysfunction, and downstream systemic inflammation, which may contribute to the development of remote organ injury.^4,5,11, 12, 13 Therefore, strategies that attenuate early intestinal injury during aortic occlusion could have important translational implications, while requiring careful interpretation within the constraints of the experimental model.

Hemoglobin vesicles (HbVs) are liposome-encapsulated hemoglobin-based oxygen carriers developed as artificial red blood cells.14, 15, 16 Their physicochemical properties have been characterized in preclinical investigations.16, 17, 18 HbVs have demonstrated feasibility and potential benefit in hemorrhagic shock models,19, 20, 21 and a first-in-human phase 1 trial has been conducted.²² These features motivated us to test whether targeted intra-arterial HbV delivery during aortic cross-clamping could attenuate early intestinal I/R injury.

We hypothesized that intra-arterial HbV delivery during thoracic aortic cross-clamping may reduce early intestinal injury. This reductionist, hypothesis-generating proof-of-principle study was designed to detect large physiological/histological effects, not subtle mechanistic differences.

Methods

HbV preparation

HbVs were prepared under aseptic conditions as previously described.^14,15 Hemoglobin purified from outdated human blood donations was encapsulated into liposomes and suspended in saline at approximately 10 g/dL. The mean particle diameter was 251 ± 80 nm, and oxygen affinity was 15 Torr.¹⁶

Animals and experimental design

All procedures were approved by the Institutional Animal Care and Use Committee of Nara Medical University and complied with the ARRIVE guidelines. Male Wistar rats aged 8 to 9 weeks (370-400 g) were used to reduce variability from hormonal cycling and to maintain consistency with prior HbV studies. Animals were assigned to three groups using a random number table: HbV group (n = 5), saline control group (n = 5), or sham group (n = 3). Outcome assessments were conducted without blinding, which is recognized as a limitation of the present study. An overview of the experimental protocol is shown in Fig 1, A, and the timing of physiological measurements and tissue sampling is summarized in Fig 1, B.

Fig 1.

Experimental setup and protocol for thoracic aortic cross-clamping and intra-arterial treatment in a rat model. (A) Schematic overview of the instrumentation, thoracic aortic cross-clamping (60 minutes), and reperfusion (60 minutes) procedures. Femoral arterial pressure was monitored continuously through the femoral arterial catheter and decreased to <5 mm Hg immediately after aortic cross-clamping, which was sustained throughout the cross-clamp period, confirming profound hypotension and ischemia distal to the aortic cross-clamp. (B) Study timeline depicting the timing of intra-arterial administration (HbV 1.25 mL/kg or equal-volume saline every 10 minutes during cross-clamping), physiological measurements (baseline and postreperfusion), blood sampling for lactate, and tissue harvest at 60 minutes after reperfusion. FiO₂, fraction of inspired oxygen; HbV, hemoglobin vesicle; HE, hematoxylin and eosin; iNOS, inducible nitric oxide synthase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Thoracic aortic cross-clamping model

Under isoflurane anesthesia and mechanical ventilation (FiO₂ 0.21), a left thoracotomy was performed and the descending thoracic aorta was cross-clamped for 60 minutes, followed by 60 minutes of reperfusion, based on a previous model identifying the longest tolerated occlusion time.⁹ Femoral arterial pressure was continuously monitored through the femoral arterial catheter and was found to decrease to <5 mm Hg immediately after aortic cross-clamping. This was sustained throughout the cross-clamp period, confirming profound hypotension and ischemia distal to the aortic cross-clamp.

Intra-arterial administration protocol

HbV (1.25 mL/kg) or saline was administered intra-arterially via the femoral artery every 10 minutes during cross-clamping. This intermittent bolus regimen was selected to owing to feasibility during occlusion workflows while limiting continuous volume loading; dose optimization remains an objective for future studies.19, 20, 21 Sham animals underwent instrumentation without cross-clamping.

Outcomes and tissue collection

The primary outcomes included systolic blood pressure (SBP) measured at baseline (preocclusion) and after reperfusion (postreperfusion), plasma lactate concentration after reperfusion, histological injury (Chiu score), and quantitative markers of apoptosis and inflammation (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]-positive area fraction [%] and inducible nitric oxide synthase [iNOS]-positive area fraction [%]).²³ Intestinal tissue was harvested 60 minutes after reperfusion for histological analyses.

Statistical analysis

Given the small sample size, nonparametric tests were performed. The Mann-Whitney U test was used for two-group comparisons, and the Kruskal-Wallis test with Bonferroni-adjusted pairwise testing were used for three-group comparisons. A two-tailed P value of <.05 was considered statistically significant.

Results

All animals completed the experimental protocol without procedural complications.

The SBP differed significantly among groups after reperfusion (Kruskal-Wallis, P = .005). HbV-treated rats maintained higher postreperfusion SBP compared with saline controls (56 ± 4 mm Hg vs 34 ± 8 mm Hg; P = .002), whereas the baseline (preocclusion) SBP was comparable among groups (Fig 2). Furthermore, plasma lactate levels differed among groups after reperfusion (Kruskal-Wallis, P = .005), with lower concentrations in the HbV group compared with those in the controls (2.7 ± 0.4 mmol/L vs 7.5 ± 0.4 mmol/L; P < .001) (Fig 2).

Fig 2.

Baseline and postreperfusion systolic blood pressure (SBP) and plasma lactate levels SBP measured at baseline (pre-occlusion) and 60 minutes after reperfusion (postreperfusion), and plasma lactate concentration at 60 minutes after reperfusion are shown for sham (n = 3), control (n = 5), and HbV (n = 5) groups. Data are expressed as the mean ± standard deviation. HbV, hemoglobin vesicle.

Histological injury was attenuated in HbV-treated rats. Chiu scores were lower in the HbV group compared with those in controls (median 2.0 vs 4.0; P = .018), whereas sham animals showed no mucosal injury (Fig 3).

Fig 3.

Histological assessment of intestinal mucosal injury using the Chiu score. Chiu scores were lower in the HbV group than in saline controls (median 2.0 vs 4.0; P = .018). Sham animals showed no mucosal injury. Data are presented as the median (interquartile range). HbV, hemoglobin vesicle.

Apoptotic and inflammatory marker levels were similarly reduced. The TUNEL-positive area fraction (%) in the HbV group was lower than in the controls (0.09 ± 0.01% vs 0.23 ± 0.05%; P = .003), as was the iNOS-positive area fraction (%) (0.10 ± 0.04% vs 0.27 ± 0.08%; P = .006) (Fig 4). Representative histological findings are shown in Fig 5.

Fig 4.

Quantitative analysis of TUNEL and iNOS positivity in the small intestine after ischemia-reperfusion (I/R). (A) TUNEL-positive area fraction (%) and (B) iNOS-positive area fraction (%) for the saline control and HbV groups at 60 minutes after reperfusion are shown. HbV treatment significantly reduced TUNEL positivity (P = .003) and iNOS positivity (P = .006) compared with that in saline controls. HbV, hemoglobin vesicle; iNOS, inducible nitric oxide synthase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Fig 5.

Representative histological findings after thoracic aortic cross-clamping and reperfusion. Representative images of HE staining, TUNEL staining, and immunohistochemical staining for iNOS in sham, saline control, and HbV-treated animals. The images illustrate reduced epithelial injury, apoptosis, and iNOS expression in HbV-treated rats compared with that in the controls. HbV, hemoglobin vesicle; HE, hematoxylin and eosin; iNOS, inducible nitric oxide synthase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Discussion

The findings of this reductionist proof-of-principle study suggests that targeted intra-arterial HbV administration during thoracic aortic cross-clamping can attenuate early intestinal I/R injury. Compared with saline controls, HbV-treated animals exhibited higher postreperfusion SBPs, lower lactate levels, reduced histopathological injury, and decreased TUNEL and iNOS positivity.

The conceptual novelty of this work lies in the targeted intra-arterial application of an oxygen carrier during complete aortic cross-clamping. Unlike strategies that mitigate ischemic burden by modulating occlusion completeness or duration,1, 2, 3^,8, 9, 10 this strategy is intended to augment distal oxygen-carrying capacity downstream of the clamp. The findings of HbV characteristics and prior preclinical studies support the feasibility of this approach.14, 15, 16, 17, 18, 19, 20, 21

Regarding mechanistic findings, the present study assessed downstream injury marker levels (TUNEL and iNOS), but did not directly measure mesenteric perfusion, tissue oxygen tension, oxidative stress, or inflammatory mediators. Accordingly, enhanced oxygen delivery is plausible but not proven, and the involvement of other mechanisms—such as altered vascular tone or inflammatory signaling—cannot be excluded. Direct perfusion/oxygenation measurements and biodistribution tracking of HbV during cross-clamping should be incorporated into future studies. Importantly, preferential delivery of HbV to ischemic bowel was not directly assessed.

Improved postreperfusion SBP should not be interpreted as definitive evidence of enhanced microvascular oxygenation. Alternative explanations, including volume-related effects or altered vascular tone, may also contribute to this phenomenon.

This study has limitations. It used a small animal nonhemorrhagic model and focused on early (60 minutes) reperfusion injury. Mechanistic assessments were limited, and outcome evaluation was not fully blinded. The sample size was designed to detect large effects and the findings should be interpreted as hypothesis generating. Finally, only male animals were used; therefore, sex-dependent effects cannot be excluded.

Overall, these findings support the feasibility of targeted intra-arterial oxygen carrier delivery as a potential adjunctive approach to mitigate early distal organ injury during aortic cross-clamping, warranting mechanistic and safety-focused follow-up studies in more clinically representative hemorrhagic shock settings.

Conclusions

In this exploratory proof-of-principle rat model, intra-arterial HbV administration during thoracic aortic cross-clamping attenuated early intestinal I/R injury, indicated by reduced histopathological injury, apoptosis, and iNOS expression. Thus, HbVs may represent a potential adjunctive approach to protect distal organs during prolonged aortic occlusion.

The authors thank Dr. Tomoko Kure and Naoko Kobayashi, Department of Chemistry, Nara Medical University, for the preparation of HbV.

Data availability statement

The data that support the findings of this study are available from the corresponding author, K.T., upon reasonable request.

Funding

This research was partly supported by the Japan Agency for Medical Research and Development (AMED) (grant numbers: JP21ym126034 and JP24ym012654) and the Japan Society for the Promotion of Science (JSPS) (grant number: 24K12206).

Disclosures

The authors are inventors of patented products related to the production and utilization of HbVs.