Transamniotic Stem Cell Therapy (TRASCET) Modulates Uterine Natural Killer Cell (uNK) Activity in the Hypoxia Model of Intrauterine Growth Restriction (IUGR).

Intrauterine Growth Restriction (IUGR) pathophysiology is driven by abnormal uterine natural killer cell (uNK) activity leading to placental dysfunction. Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) can improve experimental IUGR by mechanisms not fully understood. We sought to examine TRASCET's effects in downstream products of uNKs in a model of IUGR. Fifteen Sprague-Dawley dams were exposed to alternating hypoxia (10.5% O2) from gestational-day 15 (E15) until term (E21). Their fetuses (n=189) were divided into 4 groups. One group remained untreated (n=52), while three groups received volume-matched intra-amniotic injections of either saline (sham, n=44), or a suspension of amniotic fluid-derived MSCs, either in their native state (TRASCET, n=50) or "primed" to an enhanced anti-inflammatory phenotype (TRASCET-Primed, n=43). Normal fetuses served as controls (n=33). At term, various analyses were performed, including ELISA for surrogates of placental inflammation and uNK activity. Statistical comparisons included Bonferroni-adjusted criterion. Overall survival from hypoxia was 74% (140/189). Placental efficiency was lower in untreated and sham but normalized in both TRASCET groups (p<0.001-0.469). Interleukin-17, a stimulator of uNK cells, was elevated from normal in all groups (p<0.001 for all). Interferon-gamma, released from activated uNK cells, was elevated in all groups except sham, but lower than the untreated in both TRASCET groups (p=<0.001-0.062). Tumor necrosis factor-alpha, also produced by uNKs, was elevated in untreated and sham (p<0.001 for both), but normalized by TRASCET (p=0.054) and even lowered from normal in TRASCET-Primed (p<0.001). Vascular endothelial growth factor, also released by uNKs, was elevated in untreated and sham but lower than normal in both TRASCET groups (p<0.001 for all). We conclude that TRASCET with MSCs modulates the activity of placental uNK cells in experimental IUGR, with distinct effects on their downstream products. This mechanistic insight may inform the development of novel strategies for the management of this disease.

Comparative Effects on Fetal Hematopoiesis and Placental Inflammation From Mesenchymal and Hematopoietic Stem Cells as Agents of Transamniotic Stem Cell Therapy (TRASCET) in a Syngeneic Model of Intrauterine Growth Restriction.

PURPOSE: We compared transamniotic stem cell therapy (TRASCET) using either mesenchymal (MSCs) or hematopoietic (HSCs) stem cells on fetal hematopoiesis in a syngeneic model of intrauterine growth restriction (IUGR). METHODS: Lewis dams exposed to cycling hypoxia (10.5% O2) in late gestation had their fetuses (n = 83) either receiving no intervention (untreated; n = 9), or intra-amniotic injections of either HSCs (HSC; n = 34), MSCs primed to an enhanced anti-inflammatory phenotype (primed-MSC; n = 28), or saline (sham; n = 12). Normal controls (n = 18) were also studied. Complete peripheral blood counts and placental ELISA for inflammation and angiogenesis markers were performed at term. RESULTS: Overall survival from hypoxia was 41% (34/83). Red blood count (RBC), hematocrit (Hct) and hemoglobin levels (Hb) were all significantly decreased from normal in all hypoxia groups. TRASCET with primed-MSC had significantly higher RBC, Hct, and Hb levels than sham (p = 0.01-0.03, pairwise), though not than untreated (which had no surgical blood loss). The HSC group had only significantly higher Hb levels than sham (p = 0.005). TRASCET with primed-MSC had significantly lower levels of placental TNF-α than sham (p = 0.04), but not untreated. CONCLUSIONS: MCSs seem more effective than HSCs in enhancing hematopoiesis when used as donor cells for TRASCET in a syngeneic model of IUGR. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Bidirectional Feto-Maternal Traffic of Donor Mesenchymal Stem Cells Following Transamniotic Stem Cell Therapy (TRASCET).

PURPOSE: Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) has emerged experimentally as a potential treatment for different congenital diseases and maternal diseases of pregnancy. The broad applicability of TRASCET is predicated on hematogenous routing of donor MSCs via the placenta. We investigated whether donor MSC kinetics includes bidirectional traffic between the fetus and mother. METHODS: Eight time-dated dams had their fetuses (n = 96) divided in 4 groups on gestational day 17 (E17, term = E21). Groups populating one uterine horn received intra-amniotic injections (50 µL) of either donor amniotic fluid-derived MSCs (2×106 cells/mL) labelled with a firefly luciferase reporter gene (MSC-injected, n = 32), or of acellular luciferase (luciferase-injected, n = 26). Contra-lateral (CL) horn fetuses received no injection (MSC-CL, n = 20 and luciferase-CL, n = 18). At term, samples from 11 fetal anatomical sites from CL fetuses, along with placentas from all fetuses and maternal blood were screened for luciferase activity via microplate luminometry. RESULTS: Overall survival was 95 % (91/96). When controlled by the acellular injection, positive luciferase activity was observed in the placentas of all MSC-injected fetuses, confirming viability of the donor cells at term. When controlled by the acellular injection group, MSC-CL fetuses showed positive luciferase activity in the bone marrow, peripheral blood, brain and skin (p = <0.001-0.048). No luciferase activity was detected in any maternal blood sample. CONCLUSION: Amniotic fluid-derived MSCs can traffic between the fetus and mother in both directions after simple intra-amniotic injection, in a healthy rat model. This phenomenon must be considered in TRASCET performed in twin/multiple pregnancies. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

A Medium-Chain Fatty Acid Analogue Prevents Intestinal Failure-Associated Liver Disease in Preterm Yorkshire Piglets.

BACKGROUND & AIMS: At least 20%-30% of patients with intestinal failure receiving long-term parenteral nutrition will develop intestinal failure-associated liver disease (IFALD), for which there are few therapeutic options. SEFA-6179 is a first-in-class structurally engineered medium-chain fatty acid analogue that acts through GPR84, PPARα, and PPARγ agonism. We hypothesized that SEFA-6179 would prevent biochemical and histologic liver injury in a preterm piglet model of IFALD. METHODS: Preterm Yorkshire piglets were delivered by cesarean section, and parenteral nutrition was provided for 14 days via implanted central venous catheters. Animals were treated with either medium-chain triglyceride vehicle control or SEFA-6179. RESULTS: Compared to medium-chain triglyceride vehicle at day of life 15, SEFA-6179 prevented biochemical cholestasis (direct bilirubin: 1.9 vs <0.2 mg/dL, P = .01; total bilirubin: 2.7 vs 0.4 mg/dL, P = .02; gamma glutamyl transferase: 172 vs 30 U/L, P = .01). SEFA-6179 also prevented steatosis (45.6 vs 13.9 mg triglycerides/g liver tissue, P = .009), reduced bile duct proliferation (1.6% vs 0.5% area cytokeratin 7 positive, P = .009), and reduced fibrosis assessed by a masked pathologist (median Ishak score: 3 vs 1, P = 0.007). RNA sequencing of liver tissue demonstrated that SEFA-6179 broadly impacted inflammatory, metabolic, and fibrotic pathways, consistent with its in vitro receptor activity (GPR84/PPARα/PPARγ agonist). CONCLUSIONS: In a preterm piglet model of IFALD, SEFA-6179 treatment prevented biochemical cholestasis and steatosis and reduced bile duct proliferation and fibrosis. SEFA-6179 is a promising first-in-class therapy for the prevention and treatment of IFALD that will be investigated in an upcoming phase II clinical trial.

Morphometric, Developmental, and Anti-Inflammatory Effects of Transamniotic Stem Cell Therapy (TRASCET) on the Fetal Heart and Lungs in a Model of Intrauterine Growth Restriction.

Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) can attenuate placental inflammation and minimize intrauterine growth restriction (IUGR). We sought to determine whether MSC-based TRASCET could mitigate fetal cardiopulmonary effects of IUGR. Pregnant Sprague-Dawley dams were exposed to alternating 12-h hypoxia (10.5% O2) cycles in the last fourth of gestation. Their fetuses (n = 155) were divided into 4 groups. One group remained untreated (n = 42), while three groups received volume-matched intra-amniotic injections of either saline (sham; n = 34), or of syngeneic amniotic fluid-derived MSCs, either in their native state (TRASCET; n = 36) or "primed" by exposure to interferon-gamma and interleukin-1beta before administration in vivo (TRASCET-primed; n = 43). Normal fetuses served as additional controls (n = 30). Multiple morphometric and biochemical analyses were performed at term for select markers of cardiopulmonary development and inflammation previously shown to be affected by IUGR. Among survivors (75%; 117/155), fetal heart-to-body weight ratio was increased in both the sham and untreated groups (P < 0.001 for both) but normalized in the TRASCET and TRASCET-primed groups (P = 0.275, 0.069, respectively). Cardiac b-type natriuretic peptide levels were increased in all hypoxia groups compared with normal (P < 0.001), but significantly decreased from sham and untreated in both TRASCET groups (P < 0.0001-0.005). Heart tumor necrosis factor-alpha levels were significantly elevated in sham and TRASCET groups (P = 0.009, 0.002), but normalized in the untreated and TRASCET-primed groups (P = 0.256, 0.456). Lung transforming growth factor-beta levels were significantly increased in both sham and untreated groups (P < 0.001, 0.003), but normalized in both TRASCET groups (P = 0.567, 0.303). Similarly, lung endothelin-1 levels were elevated in sham and untreated groups (P < 0.001 for both), but normalized in both TRASCET groups (P = 0.367, 0.928). We conclude that TRASCET with MSCs decreases markers of fetal cardiac strain, insufficiency, and inflammation, as well as of pulmonary fibrosis and hypertension in the rodent model of IUGR.

Fetal Secretory IgA Delivery via Transamniotic Fetal Immunotherapy (TRAFIT) in a Rodent Model.

PURPOSE: We sought to determine the feasibility and routing kinetics of transamniotic fetal delivery of secretory immunoglobulin-A (SIgA), in a rodent model. METHODS: Fetuses (n = 94) from seven time-dated pregnant dams received intra-amniotic injections on gestational day 17 (E17, term = E21-22) of either saline (n = 15) or a solution of 1 mg/mL of ≥95% homogeneous human SIgA (n = 79). Animals were euthanized daily at E18-E21 for quantification of the IgA component by ELISA at gestational membranes, placenta, and select fetal anatomical sites against saline controls procured at term. Statistical analysis was by Mann-Whitney U-test. RESULTS: None of the saline-injected animals had detectable human IgA. SIgA-injected fetuses showed human IgA in the stomach aspirate, intestinal wall, lungs, liver, and serum at all time points. IgA levels were significantly higher in the gastric aspirate and in the intestine than in all other sites (p < 0.001 for both), with intestinal levels remaining stable through E18-E21 (p = 0.09-0.62 pairwise). Serum and placental levels were consistently low throughout, reaching near zero levels by E21. CONCLUSIONS: The chronology of exogenous secretory-IgA kinetics after intra-amniotic injection is suggestive of fetal uptake by ingestion, leading to consistent levels in the gastrointestinal tract. Transamniotic fetal immunotherapy (TRAFIT) with secretory-IgA may become a novel strategy for enhancing early mucosal immunity. LEVEL OF EVIDENCE: N/A (animal and laboratory study). TYPE OF STUDY: Animal and laboratory study.

Hematogenous Routing of Exogenous Messenger RNA Delivered Into the Amniotic Fluid.

INTRODUCTION: Therapies based on exogenous messenger RNA (mRNA) administration have emerged as a powerful novel strategy for the actual or potential treatment of an assortment of diseases, including congenital surgical pathologies. We sought to determine whether the minimally invasive transamniotic route could be an alternative for prenatal mRNA delivery. METHODS: Pregnant Sprague-Dawley dams underwent laparotomy followed by volume-matched intra-amniotic injections in all their fetuses (n = 120) of either a suspension of a custom firefly luciferase mRNA encapsulated by a lipid- and synthetic cationic polymer-based composite, or of a suspension of the same encapsulation components without mRNA, on gestational day 17 (E17; term = E21-22). On E18, E19, E20, and E21, samples from 14 fetal anatomical sites and maternal serum were procured for the screening of mRNA incorporation by host cells by measurement of luciferase activity via microplate luminometry. Statistical analysis was by Mann-Whitney U-test, including Bonferroni-adjustment. RESULTS: Overall survival was 87.5% (105/120). Controlled by the encapsulating composite without mRNA, luciferase activity was detected in the animals that received encapsulated mRNA in the following fetal annexes: amniotic fluid, amnion, chorion, umbilical cord, and placenta (P = 0.033 to <0.001), as well as in the following fetal sites: liver, stomach, intestines, and lungs (P = 0.043-0.002). CONCLUSIONS: Packaged exogenous mRNA can be incorporated by the fetus at least at select anatomical sites after simple intra-amniotic administration in a rodent model. The pattern and chronology of mRNA incorporation are compatible with transplacental hematogenous routing, as well as with fetal swallowing/aspiration. Further study of transamniotic mRNA administration is warranted.

Transamniotic stem cell therapy (TRASCET): An emerging minimally invasive strategy for intrauterine stem cell delivery.

Transamniotic stem cell therapy (TRASCET) is an emerging strategy for prenatal stem cell therapy involving the least invasive method described to date of delivering select stem cells to virtually any anatomical site in the fetus, including the blood and bone marrow, as well as to fetal annexes, including the placenta. Such broad therapeutic potential derives, to a large extent, from unique routing patterns following stem cell delivery into the amniotic fluid, which have commonalities with naturally occurring fetal cell kinetics. First reported experimentally only less than a decade ago, TRASCET has yet to be attempted clinically, though a first clinical trial appears imminent. Despite significant experimental advances, much promise and perhaps excessive publicity, most cell-based therapies have yet to deliver meaningful large-scale impact to patient care. The few exceptions typically consist of therapies based on the amplification of the normal biological role played by the given cells in their natural environment. Therein lays much of the appeal of TRASCET, in that it, too, is in essence a magnification of naturally occurring processes in the distinctive environment of the maternal-fetal unit. As much as fetal stem cells possess unique characteristics compared with other stem cells, so does the fetus when compared with any other age group, converging into a scenario that enables therapeutic paradigms exclusive to prenatal life. This review summarizes the diversity of applications and biological responses associated with the TRASCET principle.

A Digestive Cartridge Reduces Parenteral Nutrition Dependence and Increases Bowel Growth in a Piglet Short Bowel Model.

OBJECTIVE: To determine whether the use of an immobilized lipase cartridge (ILC) to hydrolyze fats in enteral nutrition (EN) reduces parenteral nutrition (PN) dependence in a porcine model of short bowel syndrome with intestinal failure (SBS-IF). BACKGROUND: SBS-IF occurs after intestinal loss resulting in malabsorption and PN dependence. Limited therapeutic options are available for achieving enteral autonomy. METHODS: Eleven Yorkshire piglets underwent 75% jejunoileal resection and were randomized into control (n=6) and treatment (n = 5) groups. PN was initiated postoperatively and reduced as EN advanced if predefined clinical criteria were fulfilled. Animals were studied for 14 days and changes in PN/EN calories were assessed. Intestinal adaptation, absorption, and nutrition were evaluated at the end of the study (day 15). Comparisons between groups were performed using analysis of covariance adjusted for baseline. RESULTS: ILC animals demonstrated a 19% greater reduction in PN calories ( P < 0.0001) and higher mean EN advancement (66% vs 47% of total calories, P < 0.0001) during the 14-day experiment. Treatment animals had increased intestinal length (19.5 vs 0.7%, P =0.03) and 1.9-fold higher crypt cell proliferation ( P =0.02) compared with controls. By day 15, ILC treatment resulted in higher plasma concentrations of glucagon-like peptide-2 ( P = 0.02), eicosapentaenoic acid ( P < 0.0001), docosahexaenoic acid ( P = 0.004), vitamin A ( P = 0.02), low-density lipoprotein ( P = 0.02), and high-density lipoprotein ( P = 0.04). There were no differences in liver enzymes or total bilirubin between the two groups. CONCLUSIONS: ILC use in conjunction with enteral feeding reduced PN dependence, improved nutrient absorption, and increased bowel growth in a porcine SBS-IF model. These results support a potential role for the ILC in clinical SBS-IF.

Fetal Alloimmune Hemolytic Anemia (AHA) as a Potential Target for Transamniotic Fetal Immunotherapy (TRAFIT).

PURPOSE: Fetal alloimmune hemolytic anemia (AHA) resulting from maternal antibodies against fetal erythrocytes may require fetal administration of immunoglobulin-G (IgG) via invasive methods. IgG can reach the fetal circulation after transamniotic fetal immunotherapy (TRAFIT). We sought to both develop a model of AHA and to test TRAFIT as a potential treatment. METHODS: Sprague-Dawley fetuses (n = 113) received intra-amniotic injections on gestational-day 18 (E18, term = E21) of either saline (control; n = 40), anti-rat-erythrocyte antibodies (AHA; n = 37), or anti-rat-erythrocyte antibodies plus IgG (AHA + IgG; n = 36). At term, blood was procured for red blood count (RBC), hematocrit, or ELISA for inflammatory markers. RESULTS: There was no difference in survival [95% (107/113)] across groups (p = 0.87). Both hematocrit and RBC were significantly lower in the AHA group than controls (p < 0.001). Although still significantly lower than controls (p < 0.001), both hematocrit and RBC significantly increased in AHA + IgG group compared to AHA alone (p < 0.001). Pro-inflammatory TNF-α and IL1-ß were significantly elevated from controls in the AHA group, but not in AHA + IgG (p < 0.001-0.159). CONCLUSIONS: Intra-amniotic injection of anti-rat-erythrocyte antibodies can reproduce manifestations of fetal AHA, constituting a practical model of this disease. Transamniotic fetal immunotherapy with IgG reduces anemia in this model and may emerge as a new minimally invasive means of treatment. TYPE OF STUDY: Animal and laboratory study. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Brain protection by transamniotic stem cell therapy (TRASCET) in a model of intrauterine growth restriction (IUGR).

PURPOSE: Transamniotic stem cell therapy (TRASCET) with mesenchymal stem cells (MSCs) has been shown experimentally to reverse some of the effects of intrauterine growth restriction (IUGR), apparently by attenuating placental inflammation. Neurodevelopmental deficits driven by neuroinflammation are major complications of IUGR. We sought to determine whether MSC-based TRASCET also mitigates inflammation in the fetal brain. METHODS: Pregnant Sprague-Dawley dams (n = 8) were exposed to alternating 12-hour hypoxia (10.5% O2) cycles from gestational day 15 (E15) until term (E21). One group remained untreated (n = 28 fetuses). Three groups received volume-matched intra-amniotic injections into all fetuses (n = 72) of either saline (sham; n = 19), or a suspension of amniotic fluid-derived MSCs, either in native state (TRASCET; n = 20), or primed by exposure to interferon-gamma (IFN-γ) and interleukin-1beta (IL-1ß) for 24 h prior to administration in vivo (TRASCET-Primed; n = 29). Donor MSCs were syngeneic Lewis rat cells phenotyped by flow cytometry. Normal fetuses served as controls (n = 20). Multiple analyses were performed at term, including ELISA in fetal brains for the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and IL-1ß. Statistical comparisons were by Wilcox-rank sum test, including Bonferroni-adjusted significance. RESULTS: Overall survival was 75% (88/116). Gross brain weights were significantly decreased from normal in both the untreated and sham groups (both p<0.001) and significantly increased in both TRASCET groups when compared to untreated and sham (p = 0.003 to <0.001). TRASCET-Primed led to significantly lower levels of TNF-α and IL-1ß compared to untreated (both p<0.001) and sham (p = 0.017 and p = 0.011, respectively). Non-primed TRASCET led to significantly lower levels of TNF-α and IL-1ß compared to untreated (p = 0.009 to <0.001), but not sham (p = 0.133 and p = 0.973, respectively). CONCLUSIONS: Transamniotic stem cell therapy with primed mesenchymal stem cells reverses some of the central nervous system effects of intrauterine growth restriction in a rat model, possibly by modulating neuroinflammation. TYPE OF STUDY: Animal and laboratory study. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Impact of bowel rotation and fixation on obstructive complications in congenital diaphragmatic hernia.

AIM OF THE STUDY: Small bowel obstruction (SBO) is a known complication after congenital diaphragmatic hernia (CDH) repair, which can require surgery and even extensive bowel resection causing short bowel syndrome (SBS). We investigate whether specific bowel rotation and fixation can be used as a predictor for SBO including volvulus. METHODS: A retrospective review of 256 CDH survivors following repair from 2003 to 2020 was performed. Operative notes and upper gastrointestinal series (UGI) were screened to determine the rotation and fixation of the bowel. Primary outcomes included SBO occurrence, SBO treated surgically, and volvulus. For statistical analysis Fisher's exact test was utilized. RESULTS: Twenty-two (9%) patients presented with SBO and majority, 19 (86%), required surgery. Adhesion were observed in 10 (45%), recurrence in 5 (23%), and extensive volvulus leading to SBS in 3 (14%). Both rotation and fixation were recorded in 117 (46%). Presence of left CDH with malrotation and nonfixation was a significant predictor for SBO requiring surgery (P<0.05 vs all other groups). All 3 patients with extensive volvulus had left CDH with nonfixed bowel (100%), however only 1 had malrotation (33%). CONCLUSIONS: Malrotation and nonfixation are associated with increased SBO in CDH. Normal rotation is not protective and patients are still at risk for volvulus resulting in SBS. SBO requiring surgical intervention is common in CDH. Bowel rotation and fixation are important determinants that, should be routinely documented and education about the risk of SBO should be included in family counseling. LEVEL OF EVIDENCE: Level IV - Case Series.

Transamniotic stem cell therapy (TRASCET) for intrauterine growth restriction (IUGR): A comparison between placental and amniotic fluid donor mesenchymal stem cells.

PURPOSE: Transamniotic stem cell therapy (TRASCET) with donor mesenchymal stem cells (MSCs) has been shown experimentally to reverse central effects of intrauterine growth restriction (IUGR). We sought to compare amniotic-fluid and placenta-derived MSCs (afMSCs and pMSCs, respectively) as TRASCET donor cells in a murine IUGR model. METHODS: Pregnant Sprague-Dawley dams (n=8) were exposed to alternating 12-hour hypoxia (10.5% O2) cycles, starting on gestational day 15 (E15; term=E21-22). On E17, fetuses (n=100) were divided into four groups. An untreated group had no further manipulations (n=24). Three groups received volume-matched intra-amniotic injections of either saline (sham; n=27), or suspensions of afMSCs (n=24), or pMSCs (n=25). Normal fetuses served as controls (n=21). All infused MSCs consisted of syngeneic Lewis rat cells phenotyped by flow cytometry and GFP-labeled. At term, fetal and placental morphometrics were calculated, and placental TNF-α levels were determined by ELISA. Statistical comparisons were by Fischer's T-test or Wilcoxon rank sum test (p≤0.05). RESULTS: Overall survival of the hypoxic groups was 83% (83/100). Compared to normal, maternal-adjusted fetal weights were significantly decreased in all hypoxia groups (pairwise p<0.001), however only the afMSC group showed higher adjusted-fetal weights than sham (p<0.001). Placental efficiency was decreased in untreated, sham, and pMSC groups (p<0.001-0.056) but normalized in the afMSC group (p=0.205). Maternal-adjusted placental weights were lower than normal in all hypoxia groups (p<0.001-0.045), except for the pMSC group (p=0.387). CONCLUSIONS: Amniotic fluid-derived mesenchymal stem cells are superior to their placenta-derived counterparts in transamniotic stem cell therapy for intrauterine growth restriction in a rat model. LEVEL OF EVIDENCE: Basic/Translational science.

Mitochondrial transplantation ameliorates acute limb ischemia.

OBJECTIVE: Acute limb ischemia (ALI), the most challenging form of ischemia-reperfusion injury (IRI) in skeletal muscle tissue, leads to decreased skeletal muscle viability and limb function. Mitochondrial injury has been shown to play a key role in skeletal muscle IRI. In previous studies, we have demonstrated that mitochondrial transplantation (MT) is an efficacious therapeutic strategy to replace or to augment mitochondria damaged by IRI, allowing enhanced muscle viability and function in cardiac tissue. In this study, we investigated the efficacy of MT in a murine ALI model. METHODS: C57BL/6J mice (male, 10-12 weeks) were used in a model of ALI. Ischemia was induced by applying a tourniquet on the left hindlimb. After 2 hours of ischemia, the tourniquet was released, and reperfusion of the hindlimb was re-established; either vehicle alone (n = 15) or vehicle containing mitochondria (n = 33) was injected directly into all the muscles of the hindlimb. Mitochondria were delivered at concentrations of 1 × 106 to 1 × 109 per gram wet weight to each muscle, and the animals were allowed to recover. Sham mice received no ischemia or injections but were anesthetized for 2 hours and allowed to recover. After 24 hours of recovery, limb function was assessed by DigiGait (Mouse Specifics Inc, Boston, Mass), and animals were euthanized; the gastrocnemius, soleus, and vastus medialis muscles were collected for analysis. RESULTS: After 24 hours of hindlimb reperfusion, infarct size (percentage of total mass) and apoptosis were significantly decreased (P < .001, each) in the gastrocnemius, soleus, and vastus medialis muscles in MT mice compared with vehicle mice for all mitochondrial concentrations (1 × 106 to 1 × 109 per gram wet weight). DigiGait analysis at 24 hours of reperfusion showed that percentage shared stance time was significantly increased (P < .001) and stance factor was significantly decreased (P = .001) in vehicle compared with MT and sham mice. No significant differences in percentage shared stance time (P = .160) or stance factor (P = .545) were observed between MT and sham mice. CONCLUSIONS: MT ameliorates skeletal muscle injury and enhances hindlimb function in the murine model of ALI.

Delayed Transplantation of Autologous Mitochondria for Cardioprotection in a Porcine Model.

BACKGROUND: We have previously demonstrated the efficacy of mitochondrial transplantation (MT) for the treatment of ischemia-reperfusion injury (IRI). We now investigate the efficacy of delayed MT by intracoronary administration in a model of regional IRI as a strategy for cardioprotection. METHODS: Female Yorkshire pigs (40-50 kg; n = 16) underwent 30 minutes of ischemia by snaring of the left anterior descending artery, and the hearts were then reperfused for 120 minutes. At that point, vehicle only or autologous mitochondria (1 × 109 in 5 mL of vehicle) were delivered as a bolus to the left coronary ostium, followed by a further 120-minute reperfusion. RESULTS: Echocardiographic analysis demonstrated that hearts receiving delayed MT after regional IRI had enhanced ejection fraction (P = .019), fractional shortening (P = .022), and fractional area change (P = .011) at 240 minutes of reperfusion compared with the untreated pigs. At the end of reperfusion there was a difference between the groups in measures of global left ventricular (LV) function such as LV end-diastolic pressure (P = .015) and rate of rise of LV pressure (P = .021). No significant differences were found between the groups in the area at risk (P = .48). Infarct size (% area at risk) was significantly decreased in hearts receiving MT compared with hearts receiving vehicle only (P < .001). CONCLUSIONS: Delayed MT by intracoronary injection appreciably decreases myocardial infarct size, increasing regional and global myocardial function. These results suggest that this can be a viable treatment modality in IRI, thus reducing long-term morbidity and mortality in cardiac surgical patients.

Preischemic autologous mitochondrial transplantation by intracoronary injection for myocardial protection.

OBJECTIVE: To investigate preischemic intracoronary autologous mitochondrial transplantation (MT) as a therapeutic strategy for prophylactic myocardial protection in a porcine model of regional ischemia-reperfusion injury (IRI). METHODS: The left coronary artery was cannulated in Yorkshire pigs (n = 26). Mitochondria (1 × 109) or buffer (vehicle [Veh]) were delivered as a single bolus (MTS) or serially (10 injections over 60 minutes; MTSS). At 15 minutes after injection, the heart was subjected to temporary regional ischemia (RI) by snaring the left anterior descending artery. After 30 minutes of RI, the snare was released, and the heart was reperfused for 120 minutes. RESULTS: Coronary blood flow (CBF) and myocardial function were increased temporarily during the pre-RI period. Following 30 minutes of RI, MTS and MTSS hearts had significantly increased CBF that persisted throughout reperfusion (Veh vs MTS and MTSS; P = .04). MTS and MTSS showed a significantly enhanced ejection fraction (Veh vs MTS, P < .001; Veh vs MTSS, P = .04) and developed pressure (Veh vs MTS, P < .001; Veh vs MTSS, P = .03). Regional function, assessed through segmental shortening (Veh vs MTS, P = .03; Veh vs MTSS, P < .001), fractional shortening (Veh vs MTS, P < .001; Veh vs MTSS, P = .04), and strain analysis (Veh vs MTS, P = .002; Veh vs MTSS, P = .003), was also significantly improved. Although there was no difference in the area at risk between treatment groups, infarct size was significantly reduced in both MT groups (Veh vs MTS and MTSS, P < .001). CONCLUSIONS: Preischemic MT by single or serial intracoronary injections provides prophylactic myocardial protection from IRI, significantly decreasing infarct size and enhancing global and regional function.

Mitochondrial transplantation enhances murine lung viability and recovery after ischemia-reperfusion injury.

The most common cause of acute lung injury is ischemia-reperfusion injury (IRI), during which mitochondrial damage occurs. We have previously demonstrated that mitochondrial transplantation is an efficacious therapy to replace or augment mitochondria damaged by IRI, allowing for enhanced muscle viability and function in cardiac tissue. Here, we investigate the efficacy of mitochondrial transplantation in a murine lung IRI model using male C57BL/6J mice. Transient ischemia was induced by applying a microvascular clamp on the left hilum for 2 h. Upon reperfusion mice received either vehicle or vehicle-containing mitochondria either by vascular delivery (Mito V) through the pulmonary artery or by aerosol delivery (Mito Neb) via the trachea (nebulization). Sham control mice underwent thoracotomy without hilar clamping and were ventilated for 2 h before returning to the cage. After 24 h recovery, lung mechanics were assessed and lungs were collected for analysis. Our results demonstrated that at 24 h of reperfusion, dynamic compliance and inspiratory capacity were significantly increased and resistance, tissue damping, elastance, and peak inspiratory pressure (Mito V only) were significantly decreased (P < 0.05) in Mito groups as compared with their respective vehicle groups. Neutrophil infiltration, interstitial edema, and apoptosis were significantly decreased (P < 0.05) in Mito groups as compared with vehicles. No significant differences in cytokines and chemokines between groups were shown. All lung mechanics results in Mito groups except peak inspiratory pressure in Mito Neb showed no significant differences (P > 0.05) as compared with Sham. These results conclude that mitochondrial transplantation by vascular delivery or nebulization improves lung mechanics and decreases lung tissue injury.

Alloreactivity and allorecognition of syngeneic and allogeneic mitochondria.

Previously, we have demonstrated that the transplantation of autologous mitochondria is cardioprotective. No immune or autoimmune response was detectable following the single injection of autologous mitochondria. To expand the therapeutic potential and safety of mitochondrial transplantation, we now investigate the immune response to single and serial injections of syngeneic and allogeneic mitochondria delivered by intraperitoneal injection. Our results demonstrate that there is no direct or indirect, acute or chronic alloreactivity, allorecognition or damage-associated molecular pattern molecules (DAMPs) reaction to single or serial injections of either syngeneic or allogeneic mitochondria.

A Novel Biological Strategy for Myocardial Protection by Intracoronary Delivery of Mitochondria: Safety and Efficacy.

Mitochondrial dysfunction is the determinant insult of ischemia-reperfusion injury. Autologous mitochondrial transplantation involves supplying one's healthy mitochondria to the ischemic region harboring damaged mitochondria. The authors used in vivo swine to show that mitochondrial transplantation in the heart by intracoronary delivery is safe, with specific distribution to the heart, and results in significant increase in coronary blood flow, which requires intact mitochondrial viability, adenosine triphosphate production, and, in part, the activation of vascular KIR channels. Intracoronary mitochondrial delivery after temporary regional ischemia significantly improved myocardial function, perfusion, and infarct size. The authors concluded that intracoronary delivery of mitochondria is safe and efficacious therapy for myocardial ischemia-reperfusion injury.

Mitochondrial transplantation prolongs cold ischemia time in murine heart transplantation.

BACKGROUND: Cold ischemia time (CIT) causes ischemia‒reperfusion injury to the mitochondria and detrimentally effects myocardial function and tissue viability. Mitochondrial transplantation replaces damaged mitochondria and enhances myocardial function and tissue viability. Herein we investigated the efficacy of mitochondrial transplantation in enhancing graft function and viability after prolonged CIT. METHODS: Heterotopic heart transplantation was performed in C57BL/6J mice. Upon heart harvesting from C57BL/6J donors, 0.5 ml of either mitochondria (1 × 108 in respiration buffer; mitochondria group) or respiration buffer (vehicle group) was delivered antegrade to the coronary arteries via injection to the coronary ostium. The hearts were excised and preserved for 29 ± 0.3 hours in cold saline (4°C). The hearts were then heterotopically transplanted. A second injection of either mitochondria (1 × 108) or respiration buffer (vehicle) was delivered antegrade to the coronary arteries 5 minutes after transplantation. Grafts were analyzed for 24 hours. Beating score, graft function, and tissue injury were measured. RESULTS: Beating score, calculated ejection fraction, and shortening fraction were significantly enhanced (p < 0.05), whereas necrosis and neutrophil infiltration were significantly decreased (p < 0.05) in the mitochondria group as compared with the vehicle group at 24 hours of reperfusion. Transmission electron microscopy showed the presence of contraction bands in vehicle but not in mitochondria grafts. CONCLUSIONS: Mitochondrial transplantation prolongs CIT to 29 hours in the murine heart transplantation model, significantly enhances graft function, and decreases graft tissue injury. Mitochondrial transplantation may provide a means to reduce graft failure and improve transplantation outcomes after prolonged CIT.